Method and device for rapid detection of bacterial antibiotic resistance/susceptibility

ABSTRACT

Described herein is a method and a device for expediting delivery of an agent to a damaged bacterial cell. In one embodiment, the methods and devices are useful for screening candidate antibiotics. In another embodiment, the methods and devices described herein are used to determine susceptibility of bacteria to an antibiotic. The methods also provide a method for determining an appropriate antibiotic to treat an individual having a bacterial infection.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application under 35 U.S.C.§120 of U.S. Ser. No. 13/283,892, filed on Oct. 28, 2011, which is acontinuation application under 35 U.S.C. §120 of an internationalapplication No. PCT/US2010/033523, filed on May 5, 2010, which claimsthe benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.61/175,605, filed May 5, 2009, the contents of which are incorporatedherein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under Contract No.AI079474 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

BACKGROUND

Disease causing microbes that have become resistant to drug therapy arean increasing public health problem. Factors contributing to the rise inantibiotic resistance include widespread and inappropriate prescriptionof broad spectrum antibiotics and patient non-compliance to antibioticregimens. The combination of these factors has resulted in some soberingstatistics. For example, an estimated 70 percent of pathogenic bacteriain hospitals are resistant to at least one of the drugs most commonlyused to treat infections (Federal Drug Administration (2007)).Staphylococcus aureus, a well known major cause of nosocomialinfections, has recently taken on a new role in causing new cases ofcommunity-acquired infections in hosts without significant predisposingrisk factors. The number of S. aureus infections and its resistance to avariety of antibiotics is increasing with 40%-60% of nosocomial S.aureus infections in the U.S. being methicillin-resistant and many beingmulti-drug resistant.

SUMMARY OF THE INVENTION

Described herein are rapid diagnostic methods and devices fordetermining antibiotic susceptibility of bacteria in a sample to enablephysicians to accurately and rapidly treat bacterial pathogens. Thediagnostic methods are based on the observation that the application ofshear stress (and/or chemical stress) to bacteria in the presence of anantibiotic permits one to determine the sensitivity (or resistance) of abacterium to the antibiotic without requiring a cell growth phase of thebacterium. Shear and/or chemical stress applied to bacteria catalyzesthe biochemical pathways to repair damage to the cells. These pathwaysare the targets of antibiotics and therefore repair is inhibited in thepresence of the antibiotic. In general, bacteria that are susceptible tosuch an antibiotic will die in the presence of a stressor, whileresistant strains can repair the stress-induced damage. Thus, thesusceptibility of the organism can be determined without waiting forbacterial growth.

The methods and devices described herein are also useful for rapiddetermination of an appropriate antibiotic useful for treating aninfection in an individual.

In one aspect, the methods described herein relate to a method forexpediting delivery of an agent comprising a reporter moiety to adamaged cell, the method comprising: (a) immobilizing bacteria to asolid support, (b) contacting said bacteria with an agent comprising areporter moiety, (c) subjecting said immobilized bacteria to a stressorin the presence or absence of an antibiotic, and (d) detecting a signalfrom said reporter moiety, wherein if a signal is detected the agent hasbeen delivered into the cell and wherein the signal is indicative ofcell damage.

In one embodiment of this aspect and all other aspects described herein,detection is performed between 5 and 25 minutes from contacting thebacteria with the agent.

In another embodiment of this aspect and all other aspects describedherein, the chemical stress comprises lysostaphin. In anotherembodiment, the chemical stress comprises lysozyme, an endolysin,oxidative stress or a porin.

In another embodiment of this aspect and all other aspects describedherein, the physical stress comprises shear stress, osmotic stress,acidic pH or basic pH.

In another embodiment of this aspect and all other aspects describedherein, the detecting step comprises detecting fluorescence emissionfrom a fluorescent dye.

In another embodiment of this aspect and all other aspects describedherein, the agent comprises a fluorescent dye.

In another embodiment of this aspect and all other aspects describedherein, the fluorescent dye detects (e.g., stains) gram negative and/orgram positive bacteria.

In one embodiment, the fluorescent dye is non-toxic, taken up only bydamaged cells, and/or has a high signal to noise ratio.

In another embodiment of this aspect and all other aspects describedherein, the fluorescent dye is Sytox green or DiBAC₄(3).

In another embodiment of this aspect and all other aspects describedherein, the solid support comprises a glass slide.

In another embodiment of this aspect and all other aspects describedherein, the glass slide is functionalized.

In another embodiment of this aspect and all other aspects describedherein, the method is automated.

In another aspect, the methods and devices described herein relate to adevice comprising a multiple channel flow cell, each channel of whichcomprises one wall decorated with immobilized bacteria. Trapped bacteriaare exposed to shear stress by flowing fluid continuously across thebacterial surface. The fluid is a mixture of growth media andfluorescent dye with or without antibiotic. The fluorescent dye ischosen such that it only stains the cell if the membrane becomespermeabilized or the membrane potential changes indicating that thedamaged cell is unable to recover in the presence of an antibiotic.

Provided herein in one aspect is a device for determining antibioticsensitivity of bacteria, the device comprising: (a) two solid supportsseparated by a gasket, wherein a channel or a plurality of channels isformed between the two solid supports, (b) a metal housing comprising aninlet and outlet opening; and (c) a pump.

In one embodiment of this aspect and all other aspects described herein,each channel is filled with a different antibiotic.

In another embodiment of this aspect and all other aspects describedherein, each channel is filled with an agent comprising a reportermoiety that preferentially binds to damaged bacteria.

In one embodiment of this aspect and all other aspects described herein,the solid support comprises a glass slide or a plastic (e.g.,polystyrene) surface.

In another embodiment of this aspect and all other aspects describedherein, the gasket comprises silicone rubber.

In another embodiment of this aspect and all other aspects describedherein, one of the two solid supports is functionalized to permitimmobilization of a bacterial cell.

In another embodiment of this aspect and all other aspects describedherein, the device further comprises immobilized bacterial cells.

In another embodiment of this aspect and all other aspects describedherein, the channel is 50-900 μm wide.

In another embodiment of this aspect and all other aspects describedherein, the pump comprises a syringe pump.

In another embodiment of this aspect and all other aspects describedherein, the device further comprises (a) tubing that connects the pumpto the inlet opening and permits fluid to be pumped into the inletopening from the pump; and (b) tubing that connects the outlet openingto the pump and permits fluid to be returned to the pump from the outletopening.

In another embodiment of this aspect and all other aspects describedherein, the metal housing can be integrated into a microscope stage.

In another embodiment of this aspect and all other aspects describedherein, the device further comprises a microscope.

In another embodiment of this aspect and all other aspects describedherein, the device further comprises a camera.

The device of claim 1, further comprising a plurality of channels.

Also described herein is a device for determining antibiotic sensitivityof bacteria as shown in FIG. 2A.

In another aspect, provided herein is a method for determiningsensitivity of bacteria to an antibiotic, the method comprising: (a)immobilizing bacteria to a solid support, (b) contacting said bacteriawith an agent comprising a reporter moiety, which preferentially bindsto damaged bacterial cells, (c) subjecting said immobilized bacteria toa stressor in the presence or absence of an antibiotic, (d) detecting asignal from said reporter moiety, wherein detection of said signalindicates that said bacteria are susceptible to said antibiotic, andwherein a lack of signal indicates that said bacteria are resistant tosaid antibiotic.

In one embodiment of this aspect and all other aspects described herein,the stressor comprises physical and/or chemical stress.

Also provided herein is a method for treating an individual having abacterial infection, the method comprising: (a) immobilizing bacteriaobtained from a biological sample from said individual to a solidsupport, (b) contacting said bacteria with an agent comprising areporter moiety, which preferentially binds to damaged bacterial cells,(c) subjecting said immobilized bacteria to a stressor in the presenceof an antibiotic, (d) detecting a signal from said reporter moiety,wherein detection of said signal indicates that said bacteria aresusceptible to said antibiotic, and (e) administering said antibiotic tosaid individual, thereby treating said bacterial infection.

In one embodiment of this aspect and all other aspects described herein,the stressor comprises physical and/or chemical stress.

In another embodiment of this aspect and all other aspects describedherein, the method comprises subjecting the bacteria to a panel of atleast two different antibiotics.

In another embodiment of this aspect and all other aspects describedherein, the chemical stress comprises lysostaphin, lysosyme, anendolysin, oxidative stress or a porin.

In another embodiment of this aspect and all other aspects describedherein, the physical stress comprises shear stress, osmotic stress,acidic pH or basic pH.

In another embodiment of this aspect and all other aspects describedherein, the detecting step comprises detecting fluorescence.

In another embodiment of this aspect and all other aspects describedherein, the agent comprises a fluorescent dye.

In another embodiment of this aspect and all other aspects describedherein, the fluorescent dye detects (e.g., stains) gram negative and/orgram positive bacteria.

In another embodiment of this aspect and all other aspects describedherein, the fluorescent dye is Sytox green or DiBAC₄(3).

In another embodiment of this aspect and all other aspects describedherein, the solid support comprises a glass slide.

In another embodiment of this aspect and all other aspects describedherein, the glass slide is functionalized.

In another embodiment of this aspect and all other aspects describedherein, the method comprises a high-throughput method.

Also described herein is a machine for obtaining data regarding responseof bacteria to an antibiotic in a biological sample from a subjectcomprising: (a) a flow chamber assay device comprising immobilizedbacterial cells from a biological sample and a plurality of channelsthat permit contact with one or more antibiotics or mixtures thereof,and additionally allowing for a stressor to be added to the bacterialcells; (b) a determination system configured to detect entry of a testagent comprising a detectable moiety into the immobilized bacterialcells upon presence of a stressor; (c) a storage device configured tostore data output from the determination system; (d) a comparison moduleadapted to compare the data stored on the storage device with referenceand/or control data and optionally further adapted to compare theintensity of a signal from the detectable moiety among the plurality ofchannels, and (e) a display module for displaying a page of retrievedcontent for the user on a client computer, wherein (i) the retrievedcontent for each channel is detection of the presence of the detectablemoiety in the bacterial cell in the presence of an antibiotic and thesignal is that the cell is susceptible to the antibiotic, and/or (ii)the retrieved content for each channel is the absence of the detectablemoiety in the bacterial cell in the presence of an antibiotic and thesignal is that the cell is resistant to the antibiotic, and/or (iii) theretrieved content for each channel is a signal intensity of thedetectable moiety in the presence of an antibiotic among the pluralityof channels, wherein the highest intensity detected among a plurality ofdifferent channels of the device, produces a signal that the antibioticis preferred for treatment of a bacterial infection comprising thebacterial cells compared to any of the other channels, wherein a lowersignal intensity is detected.

In one embodiment of this aspect and all other aspects described herein,the control data comprises data from a channel in the device that is nottreated with an antibiotic.

In another embodiment of this aspect and all other aspects describedherein, the determination system is configured to detect fluorescenceemission data.

Also described herein is a computer system for obtaining data regardinga biological specimen comprising: (a) a determination system configuredto receive detection information, wherein the detection informationcomprises intensity of a signal from an agent comprising a detectablemoiety upon entry into an immobilized bacterial cell in the presence ofa stressor; (b) a storage device configured to store data output fromthe determination system; (c) a comparison module adapted to compare thedata stored on the storage device with reference and/or control data,and to provide a retrieved content, and (d) a display module fordisplaying a page of the retrieved content for the user, wherein theretrieved content indicates that a bacterial cell (i) is susceptible toan antibiotic, (ii) is resistant to an antibiotic, and/or (iii) is apreferred antibiotic for treatment of a bacterial infection comprisingthe bacterial cells.

Another aspect described herein relates to a computer readable mediumhaving computer readable instructions recorded thereon to definesoftware modules including a determination system and a comparisonmodule for implementing a method on a computer to determine a preferredantibiotic for treatment of a bacterial infection, the methodcomprising: (a) storing data about detection information representingentry of an agent comprising a reporter moiety into an immobilized andstressed bacterial cell or population of cells taken from a subject witha bacterial infection, wherein the detection information comprises theintensity of a signal from the agent comprising a detectable moiety uponentry into the immobilized and stressed bacterial cell; (b) comparingwith the comparison module the data stored on the storage device withreference and/or control data, and to provide a retrieved content, and(c) displaying the retrieved content for the user, wherein the absenceof entry of the agent into the bacterial cell indicates that theimmobilized and stressed bacterial cell is resistant to the antibiotic,wherein the presence of the agent in the bacterial cell indicates thatthe immobilized and stressed bacterial cell is susceptible to theantibiotic, and wherein the antibiotic having the highest intensity ofsignal from the reporter moiety in the population of cells indicatesthat the antibiotic is the preferred antibiotic for treatment of asubject having the bacterial infection comprising the bacterial cells.

Also provided herein is a method for screening a candidate antibioticfor activity against a bacterial strain, the method comprising: (a)immobilizing bacteria to a solid support, (b) contacting said bacteriawith an agent comprising a reporter moiety, which preferentially bindsto damaged bacterial cells, (c) subjecting said immobilized bacteria toa stressor in the presence or absence of an effective amount of acandidate antibiotic, (d) detecting a signal from said reporter moiety,wherein detection of the signal indicates that the candidate antibiotichas activity against the bacterial strain, and wherein lack of signalindicates that the candidate antibiotic has no activity against thebacterial strain.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic diagram depicting a functional set-up of adevice as described herein (left) and a graph depicting fluorescencemeasurements over time for antibiotic resistant and antibioticsusceptible bacterial strains (right).

FIGS. 2A-2B show an exploded view of a flow cell (100) including a flowcell housing designed to insert into a fluorescent microscope stage(2A), wherein 110 is an inlet, 120 is an outlet, 130 is a glass slidewith inlet/outlet, 140 is a silicone channel, 150 is a class slide withepoxide coating and 160 is a silicone padding. FIG. 2B shows a systemfor use with a flow cell comprising a fluorescent microscope, wherein200 is an inverted fluorescence and phase contrast microscope, 300 is asyringe side (upstream) tubing, 400 is stage/shutter controller, 500 isa 60 ml exelint syringe, 600 is a KDS 230 syringe pump, 700 is anX/Y-stage, 800 is an Z-axis stepper motor, 900 is a vibration isolatedtable (workstation) TE-CCD camera, 1000 denotes fluorescent excitation,1100 is a waste bottle, and 1200 is a waste side (downstream) tubing.2000 denoted an automatic image acquisition with XYZ-stage, shutter andpump control.

FIG. 3 shows a graph depicting cell death over time formethicillin-resistant S. aureus (MRSA) and methicillin-sensitive S.aureus (MSSA) during and following exposure to 10 μg/mL oxacillin, 0.6ng/mL lysostaphin, and ˜4200 s⁻¹ shear.

FIG. 4 shows an exemplary reaction for covalent attachment of abacterium to epoxide glass substrates, where the proteins are organicmolecules on the bacterium's surface.

FIG. 5 shows an exemplary process to create a flow cell by casting amolded rubber chip using poly-dimethylsiloxane (PDMS).

FIG. 6 shows an exemplary mold design for casting a molded rubber chipusing poly-dimethylsiloxane (PDMS). Casting mold consists of (1) moldbottom part (channels); (2) mold top part (walls); and pins (holes).Aluminum a mold material allows faster manufacturing, diamond turningwithout surface treatment, but filigree structures are easily damaged ifnot handled properly. The flatness of upper PDMS surface is specified byviscosity, surface tension, and edge effects. Basic geometrymanufactured by milling: (1) inner corners need to have radii; (2) spacebetween the channels need to be big enough for allowing the end mill topass through; (3) required parallelism of channel top and bottom surfacerequire planning of stock material; and (4) due to planning the surfacequality if defined by the planning process.

FIG. 7 shows an exemplary method for casting a molded rubber chip usingpoly-dimethylsiloxane (PDMS) and considerations with regard to PDMScasting effects. Surface qualities are defined by milling process: (1)channel top; (2) channel bottom; and (3) channel wall. Surface qualitiesare defined by fluid effects: upper PDMS surface. Critical surfacequalities: channel top (optional). Critical dimensional tolerances: (1)height of the channel (±5 μm); (2) parallelism of channel top andchannel bottom (±5 μm); and (3) width of channel (±5 μm). Edge effects:(1) capillary effect causes meniscus at mold-PDMS intersection; (2)effect occurs at pins and mold top part edge; (3) effect at mold toppart has negative effects for sealing (preventing by filling the PDMS tothe same height as the mold); (4) effect at the pins can be useful forsealing (meniscus is used like an O-ring).

FIGS. 8A-B shows an exemplary 4-channel flow cell design for use withthe methods described herein (8A), and exemplary software for automationof data collection using such a flow cell (8B).

FIG. 9A-B shows representative data from a 4-channel flow cell testingthe effect of chemical stress in the presence and absence of oxacillinin Sanger 476 methicillin sensitive cells (9A) or MW2 methicillinresistant cells (9B).

FIGS. 10A-B shows representative data from a 4-channel flow cell designtesting two bacterial strains simultaneously in the presence or absenceof lysostaphin, oxacillin or a combination thereof.

FIG. 11 shows an exemplary range of shear stress levels based on varyingthe width and height of the channels within the flow cell of the assaysystem.

FIG. 12 is a block diagram showing an exemplary system for use with themethods described herein.

FIG. 13 is a block diagram showing exemplary instructions encoded on acomputer readable medium for use with the devices and systems describedherein.

DETAILED DESCRIPTION

Described herein are devices and methods to enable clinicians toprescribe targeted antibiotic therapy immediately, instead of thecurrent practice of prescribing a broad-spectrum antibiotic initiallyand changing to targeted therapies when the antibiotic susceptibilityprofile is established. The method can be integrated with a rapidisolation technique for directly testing susceptibility of clinicalspecimens, such as by using immunomagnetic beads.

Current methods for detecting antibiotic susceptibility are based on theability of bacteria to proliferate in the presence of antibiotics, andthus these techniques are time-consuming, costly, and insensitive,particularly for the evaluation of slow-growing organisms. Describedherein are methods and devices useful as a rapid susceptibility test,which circumvents the need for a bacterial growth phase. Describedherein are methods to detect the response of small numbers of cells toantibiotics in the presence of mechanical and/or chemical stressors toobviate the time needed for growth. In general, by straining the cell,the bacterium is challenged to repair itself. The repair processes ofbacterial cells are often targets of antibiotics. If that repair processis hindered by an antibiotic, the cell will die or depolarize, which canbe monitored via e.g., fluorescence stains. This methodology offersadvantages over the current techniques by providing phenotypicinformation in an ultra-rapid time frame (e.g., 5-30 minutes) allowingphysicians to make appropriate antibiotic treatment choices sooner.

Provided herein, in one aspect, is a device for measuring antibioticresistance/susceptibility to an antibiotic. In one embodiment, a device(e.g., detection system) as described herein comprises a flow cell or amultiple channel flow cell, wherein the channel or the plurality ofchannels has one wall decorated with immobilized bacteria. Trappedbacteria are exposed to shear stress by flowing fluid continuouslyacross the bacterial surface. The fluid is a mixture of growth media anda reporter stain (e.g., a fluorescent dye) with or without antibiotic.The reporter stain is chosen such that it only stains the cell if themembrane becomes permeabilized or the membrane potential changesindicating that the antibiotic has damaged the cell. The methods anddevices described herein can be multiplexed either using multipleantibiotics or multiple organisms by the addition of more fluidicchannels. In addition, the device provides a unique platform for rapidscreening of candidate antibiotics from e.g., a library of smallmolecules. The methods and devices described herein can also be used todetermine a minimum inhibitory concentration of an antibiotic with aparticular strain of bacteria. The device and method can be integratedwith current bacterial identification methodologies (e.g. culture posttesting).

DEFINITIONS

As used herein, the phrases “antibiotic sensitivity of bacteria” or“sensitivity of bacteria to an antibiotic” reflects the ability of aparticular antibiotic to prevent repair and/or promote damage to abacterial cell. The “antibiotic sensitivity” of a bacterium can beassessed by measuring the degree of stressor-induced bacterial celldamage in the presence of an antibiotic; for example, cell damage canassessed by measuring the degree of fluorescence emitted from afluorescent dye that detects damaged bacterial cells.

As used herein the terms “reporter stain,” “agent comprising a reportermoiety” and “agent comprising a detectable moiety” are usedinterchangeably and refer to a molecule that accumulates differentiallyin damaged cells and undamaged cells, and further comprises a moietythat can be used for detection. For example, an “agent comprising areporter moiety” encompasses a fluorescent dye that is taken uppreferentially by damaged cells, while it is excluded and/or extruded byundamaged or repaired cells.

As used herein, the term “reporter moiety” or “detectable moiety” refersto a molecule, or moiety of a molecule, capable of producing adetectable signal such as e.g., fluorescence, chemiluminescence, acolorimetric signal etc.

A bacterium is considered to be “resistant” or “insensitive” to anantibiotic if the bacterium recovers from stressor-induced cell damagein the presence of an antibiotic to a substantially similar degree tothat observed in an identical bacterial strain cultured in the absenceof the antibiotic; that is a detectable signal of resistant cells in thepresence of an antibiotic (e.g., fluorescent emission) is substantiallysimilar to that of cells not treated with an antibiotic. Resistance toan antibiotic can be assessed using the methods described herein bye.g., a lack of fluorescence emission from a fluorescent dye thatpreferentially binds to damaged bacterial cells.

A bacterium is considered to be “susceptible” or “sensitive” to anantibiotic if the bacterium does not recover or is delayed fromrecovering from stressor-induced cell damage in the presence of anantibiotic. A bacterium is considered susceptible if e.g., fluorescenceemission from a fluorescent dye that accumulates in dying cells is atleast 10% higher than the fluorescence detected from the same bacteriumin the absence of an antibiotic; preferably the fluorescence is at least20% higher, at least 30% higher, at least 40% higher, at least 50%higher, at least 60% higher, at least 70% higher, at least 80% higher,at least 90% higher, at least 1-fold higher, at least 2-fold higher, atleast 5-fold higher, at least 10-fold higher, at least 100-fold higher,at least 1000-fold higher, at least 10000-fold higher or more than thefluorescence detected in the absence of an antibiotic. It iscontemplated herein that a bacterium is susceptible for more than oneantibiotic tested concurrently, thus the antibiotic that produces thehighest intensity of fluorescence in a population of cells (indicatingthe highest level of cell death) is contemplated for use in treatment ofan individual.

As used herein the term “stressor” refers to a physical/mechanical(e.g., shear stress) or chemical stress (e.g., lysostaphin, osmoticstress, pH, oxidative stress, enzymatic stress (e.g., lysozyme,endolysins), etc) that induces cell damage to an immobilized bacterium.The stressor should be strong enough to permit visualization of celldamage, for example by detecting fluorescence emission from afluorescent dye that accumulates in dying cells as described herein, butshould not be so strong as to induce substantial cell death in theabsence of an antibiotic. The stressor should permit recovery of atleast 60% of bacterial cells in the absence of an antibiotic in theculture conditions; preferably a recovery of at least 70%, at least 80%,at least 90%, at least 95%, at least 99% or even a recovery of 100%(i.e., total recovery) is achieved in the absence of an antibiotic overthe time course of the methods described herein.

As used herein, the term “cell damage” is used to refer to any celldamage that can be differentially detected using an agent comprising areporter molecule (e.g., a fluorescence agent that detects damagedcells). The term “cell damage” encompasses a non-lethal and repairableloss of e.g., bacterial cell wall integrity, such that the cell wallpermits the cellular entry of a detectable agent (e.g., fluorescent dye)that is normally excluded (or vice versa). “Cell damage” can also beassessed functionally by the activation of cellular repair enzymes, suchas those that promote peptidoglycan synthesis. The term “cell damage” isnot intended to encompass damage that results in substantial bacterialcell death (e.g., greater than 40% cell death).

As used herein the terms “functionalization”, or “functionalized” areused to describe modifications to a solid support, which permit orenhance immobilization of a bacterium. Functionalization of a solidsupport can encompass, for example attaching a protein, a polymer, alinker, a particle, a chemical group, or a combination thereof to thesolid support. Some non-limiting examples of agents useful forfunctionalization include fibronectin, fibrinogen, gelatin, collagen,epoxide-activation, elastin, among others. It is preferred that thefunctionalization permits binding of a variety of bacterial speciesand/or strains, i.e., not specific to a particular bacterial strain.

As used herein, the term “high throughput” refers to a device/system ora method for determining antibiotic resistance/susceptibility from atleast two samples simultaneously, iteratively, concurrently, orconsecutively. In one embodiment the number of samples assayedsimultaneously is in the range of 1-10000 samples inclusively; inalternate embodiments the following ranges of sample number can beassayed in the high throughput device or system: 1-5000 samplesinclusive, 1-2500, 1-1250, 1-1000, 1-500, 1-250, 1-100, 1-50, 1-25,1-10, 1-5, 7500-10000, 5000-10000, 4000-10000, 3000-10000, 2000-10000,1000-10000, 500-10000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000,inclusive. The term “high-throughput” encompasses automation of themethods described herein using e.g., robotic pippettors, roboticsamplers, robotic shakers, data processing and control software, liquidhandling devices, incubators, detectors, hand-held detectors etc. Forthe purposes of automation, it may be preferred that the number ofsamples tested at one time correspond to the number of wells in astandard plate (e.g. 6-well plate, 12-well plate, 96-well plate,384-well plate, etc.). The samples can be obtained from a plurality ofindividuals, or from a plurality of samples obtained from a singleindividual. A high-throughput system permits one to test susceptibilityof a bacterial strain to multiple antibiotics simultaneously, to testfor susceptibility to a particular antibiotic in a plurality of samples,or to test multiple doses of the same antibiotic in a sample.

As used herein the phrase “panel of at least two different antibiotics”refers to a plurality of different antibiotic compounds assessed atapproximately the same time. A “plurality” refers to at least 2different antibiotics; preferably the plurality is in the range of 2-100different antibiotics, other preferred ranges include 2-90, 2-80, 2-70,2-60, 2-50, 2-40, 2-30, 2-20, 2-12, 2-10, 2-6, 2-5, 10-50, 10-40, 10-30,10-20, 12-24, 12-36, 12-48, 12-60, 12-72, 25-50, 25-60, 25-70, 50-100,60-100, 70-100, 80-100, 90-100, among others.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Bacteria

Essentially any bacteria can be tested for antibiotic susceptibility orused in screening a candidate antibiotic using the methods and devicesdescribed herein. Particularly relevant bacteria include pathogenicbacteria that infect mammalian hosts (e.g., bovine, murine, equine,primate, feline, canine, and human hosts). In one embodiment, thebacteria is one that infects and/or causes disease in a human host.Examples of pathogenic bacteria include e.g., members of a bacterialspecies such as Bacteroides, Clostridium, Streptococcus, Staphylococcus,Pseudomonas, Haemophilus, Legionella, Mycobacterium, Escherichia,Salmonella, Shigella, Vibrio, or Listeria.

Some clinically relevant examples of pathogenic bacteria that causedisease in a human host include, but are not limited to, Bacillusanthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella aborus,Brucella canis, Brucella melitensis, Brucella suis, Campylobacterjejuni, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis,Clostridium botulinum, Clostridium difficile, Clostridium perfringens,Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis,vancomycin-resistant Enterococcus faecalis, Enterococcus faecium,Escherichia coli, enterotoxigenic Escherichia coli (ETEC),enteropathogenic Escherichia coli, E. coli O157:H7, Francisellatularensis, Haemophilus influenzae, Helicobacter pylori, Legionellapneumophila, Leptospira interrogans, Listeria monocytogenes,Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae,Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa,Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium,Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermis,Staphylococcus saprophyticus, methicillin-resistant Staphylococcusaureus (MRSA), vancomycin-resistant Staphylococcus aureus (VSA),Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcuspyogenes, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.

Solid Supports

In one embodiment of the present invention, a bacterium is immobilizedto a solid substrate. Immobilization of a bacterium permits generationof a mechanical shear stress by flowing liquid over the bacterium. Inits simplest version, a solid support comprises a glass slide to whichthe bacterium binds. In other embodiments of the invention, thebacterium is anchored to a solid support, which can comprise forexample, a magnetic particle, polymeric microsphere, filter material, orthe like, which permits the generation of mechanical shear stress.

A variety of other solid substrates can be used, including, withoutlimitation, the following: cellulose; nitrocellulose; nylon membranes;papers, fiberglass, fabrics made of synthetic materials, glass beads;polystyrene matrices; activated dextran; coated polystyrene beads;agarose; polyethylene; functionalized plastic, glass, silicon, aluminum,steel, iron, copper, nickel, and gold; tubes; wells; microtiter platesor wells; slides; discs; columns; beads; membranes; well strips; films;chips; and composites thereof. In one embodiment, a portion of thesurface of a solid substrate is coated with a chemically functionalgroup to allow for binding of the bacterium to the surface of the solidsubstrate. Solid substrates with the functional group already includedon the surface can be obtained from commercial sources. In addition, thefunctional groups may be added to the solid substrates by thepractitioner.

If a solid substrate is made of a polymer, it can be produced from,without limitation, any of the following monomers: acrylic acid;methacrylic acid; vinylacetic acid; 4-vinylbenzoic acid; itaconic acid;allyl amine; allylethylamine; 4-aminostyrene; 2-aminoethyl methacrylate;acryloyl chloride; methacryloyl chloride; chlorostyrene;dischlorostyrene; 4-hydroxystyrene; hydroxymethyl styrene; vinylbenzylalcohol; allyl alcohol; 2-hydroxyethyl methacrylate; poly(ethyleneglycol) methacrylate; and mixtures thereof, together with one of thefollowing monomers: acrylic acid; acrylamide; methacrylic acid;vinylacetic acid; 4-vinylbenzoic acid, itaconic acid; allyl amine;allylethylamine; 4-aminostyrene; 2-aminoethyl methacrylate; acryloylchloride; methacryloyl chloride; chlorostyrene; dichlorostyrene;4-hydroxystyrene; hydroxymethyl styrene; vinylbenzyl alcohol; allylalcohol; 2-hydroxyethyl methacrylate; poly(ethylene glycol)methacrylate; methyl acrylate; methyl methacrylate; ethyl acrylate;ethyl methacrylate; styrene; 1-vinylimidazole; 2-vinylpyridine;4-vinylpyridine; divinylbenzene; ethylene glycol dimethacrylate;N,N′-methylenediacrylamide; N,N′-phenylenediacrylamide;3,5-bis(acryloylamido)benzoic acid; pentaerythritol triacrylate;trimethylolpropane trimethacrylate; pentaerytrithol tetraacrylate;trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate;trimethylolpropane ethoxylate (7/3 EO/OH) triacrylate;trimethylolpropane propoxylate (1 PO/OH) triacrylate; trimethylolpropanepropoxylate (2 PO/OH) triacrylate; and mixtures thereof.

In one embodiment, bacterial cells are immobilized on a porous solidsupport. The terms “porous” or “porosity” generally refers to materialshaving a distribution of pore sizes ranging from 100 nm to 1000 μm.Particularly useful inorganic fibers and fibrous material compositionsare natural and synthetic fibers made from glass, ceramic, metal,quartz, silica, silicon, silicate, silicide, silicon carbide, siliconnitride, alumina, aluminate, aluminide, carbon, graphite, boron, borate,boride, and boron nitride. Particularly useful natural or syntheticfibers and fibrous material compositions are polymer fibers made fromaromatic polyamides, nylons, polyarylonitrile, polyesters, olefins,acrylics, cellulose, acetates, anidex, aramids, azlon, alatoesters,lyocell, spandex, melamines, modacrylic, nitrile, polybenzimidazole,polyproplylene, rayons, lyorell, sarans, vinyon, triacetate, vinyl,rayon, carbon pitch, epoxies, silicones, sol gels,polyphenylene-benzobis-ozazole, polyphenylene sulfides,polytetrafluoroethylene, teflon, and low density or high densitypolyethylene. In one embodiment, the porous solid support comprises anacid-washed silk screen.

It is preferred that a solid support permits immobilization of a widevariety of bacterial species and/or strains in a non-specific manner.This can be achieved, for example, by using species non-specificreactive chemistries (e.g., epoxides) to functionalize the solidsupport. The solid support should not induce death of the cells norshould it interfere with normal cellular processes (e.g., cell wallproduction/repair, metabolism, energy production etc.).

Bacterial Immobilization

To ensure that a bacterial cell immobilization technique can be appliedto many cell types, bacteria can be immobilized using a techniquesimilar to that used for protein and DNA immobilization the field ofmicroarrays. Bacterial cells can be covalently linked to glass slidesusing chemically activated slides as the substrate (e.g. ARRAYIT®Superepoxy 2 slides, available for ARRAYIT®, Sunnyvale, Calif.).Bacteria, such as staphylococcus, can be immobilized by adherence toepoxide-activated slides. Primary amines on the bacterial cell wallsurface act as nucleophiles, attacking epoxy groups and coupling theprotein covalently to the surface. Bacteria can be immobilized on asurface (e.g., a glass slide) using any reactive chemistry that permitsimmobilization of any bacterial cell in a species non-specific manner.

To optimize the immobilization protocol to reproducibly bind live cellsto the glass slide surface at a density that enables appropriatequantification, the number of bacteria bound to the surface can becounted under an optical microscope. To ease visualization, the cellscan be stained with a fluorescent dye such as LIVE (Molecular Probes).

Alternatively, immobilization of the bacteria can be performed viabacterial adhesins. Capture agents useful for immobilizing bacteria caninclude those to which the bacteria bind in vivo for attachment to hosttissue (e.g., fibronectin, elastin, fibrinogen etc). Additionally, tohave broad strain sensitivities, the adhesins for the capture agentsshould be highly conserved.

Shear Stress

Shear stress, in the presence or absence of chemical stress, applied tobacteria will significantly reduce the time required to observe theeffects of antibiotics on cells. This is due, in part, to the ability ofbacteria to respond to their environment and adapt to stress byactivating various biochemical pathways to cope with the stressors andchanges in environment. Shear forces can be applied to induce stress onbacteria and has been shown to cause responses in various bacterialmodel systems, (e.g., Bacillus subtilis (Sahoo, S., K. K. Rao, and G. K.Suraishkumar Biotechnol Bioeng (2006) 94(1):118-27; Sahoo, S., et al.,Biotechnol Prog (2003) 19(6):1689-96), Microbacterium lacticum (Bulut,S., et al., Appl Environ Microbiol (1999) 65(10):4464-9), Bacillusthuringiensis (Wu, W. T., et al., Appl Microbiol Biotechnol (2002)58(2):175-7), and Escherichia coli (Thomas, W. E., et al., Cell (2002)109(7):913-23)). If the shear rates are high (greater than 2000 s⁻¹) orexerted over an extended period of time (hrs), shear forces can lead tocell death (Sahoo, S., et al., Biotechnol Bioeng (2006) 94(1):118-27;Sahoo, S., et al., Biotechnol Prog (2003) 19(6):1689-96; Bulut, S., etal., Appl Environ Microbiol (1999) 65(10):4464-9). More moderate shearrates can cause down-regulation of protein secretions, as in the case ofBacillus thuringiensis (Wu, W. T., et al., Appl Microbiol Biotechnol(2002) 58(2):175-7). Interestingly, in some pathogens, bacterialadhesion to target cells can increase in the presence of increased shearstress (Thomas, W. E., et al., Cell (2002) 109(7):913-23). Theseexamples of bacterial responses to shear stress indicate that shearstress can induce bacteria to activate biochemical pathways.

It is contemplated herein that a sufficient shear force needs to beapplied to stress the cells, without causing the bacteria to be shearedoff of the immobilized capture agent and then out of optical view.Additionally, at the high shear extreme, a danger exists of outrightkilling the cells. It is well within the skill of one in the art and/orusing methods described herein to identify an appropriate level of shearstress for use with the methods and devices described herein bymonitoring cell damage induced by a stressor and the degree of recoveryof bacterial cells in the absence of an antibiotic.

The amount of shear stress can be optimized for a particular bacteriumby altering the rate or velocity of flow across the immobilizedbacteria. It is contemplated herein that different strains or species ofbacteria will have different responses to a shear stress. One of skillin the art can easily optimize the amount of shear stress necessary forthe methods described herein, for example, by increasing or decreasingthe flow velocity across cells in one or more channels and comparing thelevel of e.g., fluorescence to that detected in a control channel havinga fixed flow velocity. In one embodiment, the methods and devicesdescribed herein permit the shear stress to be modified using e.g., apump or multiple pumps to control the flow velocity.

The flow rate and velocity needed to induce the shear stress isdependent on the geometry and size of the channel. Shear stress can becalculated using the formula:

shear stress=(6*Q)/(w*ĥ2)  (Formula I)

wherein Q is the flow rate, w is the width and h is the height of thechannel of the flow cell through which the medium is pumped. In theexemplary device described herein in the Examples section, flow ratestypically range between 0.5-5 mL/min, e.g., between 0.5-4 mL/min, 0.5-3mL/min, 0.5-2 mL/min, 0.5-1 mL/min, 0.5-0.75 mL/min, etc. Table 1 showssample calculations of shear stress for different rectangular surfacegeometries (e.g., channel sizes).

Shear Rate/Shear Stress (SR) Formula for shear rate (6 * Q)/(w *h{circumflex over ( )}2) w [μm] 250 w [μm] 300 w [μm] 350 w 400 w 400[μm] [μm] h [μm] 125 h [μm] 150 h [μm] 175 h [μm] 200 h 205 [μm] Q[ml/h] SR [1/s] Q SR [1/s] Q SR [1/s] Q SR [1/s] Q SR [1/s] [ml/h][ml/h] [ml/h] [ml/h] 20 8533.333333 20 4938.271605 20 3109.815355 202083.333333 20 1982.946659 40 17066.66667 40 9876.54321 40 6219.63070940 4166.666667 40 3965.893317 60 25600 60 14814.81481 60 9329.446064 606250 60 5948.839976 80 34133.33333 80 19753.08642 80 12439.26142 808333.333333 80 7931.786635 100 42666.66667 100 24691.35802 10015549.07677 100 10416.66667 100 9914.733294 120 51200 120 29629.62963120 18658.89213 120 12500 120 11897.67995 140 59733.33333 14034567.90123 140 21768.70748 140 14583.33333 140 13880.62661 16068266.66667 160 39506.17284 160 24878.52284 160 16666.66667 16015863.57327 180 76800 180 44444.44444 180 27988.33819 180 18750 18017846.51993 200 85333.33333 200 49382.71605 200 31098.15355 20020833.33333 200 19829.46659 220 93866.66667 220 54320.98765 22034207.9689 220 22916.66667 220 21812.41325 240 102400 240 59259.25926240 37317.78426 240 25000 240 23795.3599 260 110933.3333 260 64197.53086260 40427.59961 260 27083.33333 260 25778.30656 280 119466.6667 28069135.80247 280 43537.41497 280 29166.66667 280 27761.25322 300 128000300 74074.07407 300 46647.23032 300 31250 300 29744.19988 320136533.3333 320 79012.34568 320 49757.04568 320 33333.33333 32031727.14654 340 145066.6667 340 83950.61728 340 52866.86103 34035416.66667 340 33710.0932 360 153600 360 88888.88889 360 55976.67638360 37500 360 35693.03986 380 162133.3333 380 93827.16049 38059086.49174 380 39583.33333 380 37675.98652 400 170666.6667 40098765.4321 400 62196.30709 400 41666.66667 400 39658.93317 420 179200420 103703.7037 420 65306.12245 420 43750 420 41641.87983 440187733.3333 440 108641.9753 440 68415.9378 440 45833.33333 44043624.82649 460 196266.6667 460 113580.2469 460 71525.75316 46047916.66667 460 45607.77315 480 204800 480 118518.5185 480 74635.56851480 50000 480 47590.71981 500 213333.3333 500 123456.7901 50077745.38387 500 52083.33333 500 49573.66647 40 17066.66667 40 9876.5432140 6219.630709 40 4166.666667 40 3965.893317 60 25600 60 14814.81481 609329.446064 60 6250 60 5948.839976 80 34133.33333 80 19753.08642 8012439.26142 80 8333.333333 80 7931.786635 100 42666.66667 10024691.35802 100 15549.07677 100 10416.66667 100 9914.733294

Flow Media for Generating Shear Stress

It is contemplated herein that the liquid medium used to generate a flowvelocity across an immobilized bacterium is, at the least, a minimalmedia capable of sustaining bacterial cells and permitting repair ofdamage caused by the mechanical stress. It is well within the abilitiesof one skilled in the art to choose a medium that sustains bacterialcell repair. Any standard bacterial medium, such as Mueller Hintonbroth, can be used as a flow medium. The temperature, and concentrationof the medium can also be varied to optimize conditions that are usefulfor a variety of different bacterial strains.

The flow medium also comprises a reporter stain, which is an agentcomprising a detectable moiety that is added to the medium to permitvisualization of cell damage and/or cell death. A reporter stain can beany stain that (i) accumulates preferentially in damaged or dyingbacterial cells compared to undamaged or repaired cells or (ii) isexcluded preferentially in damaged or dying cells compared to undamagedor repaired cells and that provides a detectable signal. For example, areporter stain can be a fluorescent dye that stains only damaged ordying bacterial cells and an increase in fluorescence in the presence ofan antibiotic is indicative of antibiotic-induced cell death.Alternatively, a reporter stain can accumulate in living bacterial cellsand is lost when a cell is damaged, thus a decrease or loss of thereporter stain in the presence of an antibiotic is indicative ofantibiotic-induced cell death. It is preferred that the levels of areporter stain can be measured in real time to provide rapid detectionof antibiotic susceptibility to an antibiotic. The reporter stain can bee.g., a fluorescent dye, a colorimetric agent, a luminescent agent, achemiluminescent agent, a fluorophore, a pH sensitive dye, adepolarization sensitive dye, among others. In one embodiment, thereporter stain emits within the visible range (e.g., having a wavelengthof approximately 390-750 nm). It is also contemplated herein, in oneembodiment that a plurality (e.g., at least two) of different reporterstains are used together, for example, with one agent staining dead ordying cells and the other agent staining living or repaired cells. Inthis embodiment, the ratio of the two reporter stains can be used as aquantitative or qualitative measure of dead cells (e.g., antibioticsensitive cells) to repaired cells (e.g., antibiotic resistant cells).

In one embodiment, the reporter stain used with the methods and devicesdescribed herein is a fluorescent dye. A fluorescent dye can be added tothe medium for visualizing cellular damage. The fluorescent stains canbe chosen such that they do not enter the cells unless the cell wall ispermeabilized or the membrane potential changes, and thus will notinterfere with cellular repair. In one embodiment, a fluorescentdye/stain useful with the methods and devices described herein meets thefollowing criteria: (1) effectively stains both gram-negative andgram-positive bacteria, (2) does not damage cells and is not toxic tocells, (3) can be used directly in a growth medium, and (4) does notrequire pre-treatment processing steps (e.g., fixation orcentrifugation). Two non-limiting and exemplary candidate dyes that meetthe above criteria are Sytox Green and DiBAC₄(3). Sytox green stainsnucleic acids and is an unsymmetrical cyanine dye with three positivecharges. DiBAC₄(3) is a membrane-potential sensitive dye.

Chemical Stress

Damage to the bacterial cell can also be induced through chemicalstress. This is particularly important in some cases where themechanical shear forces generated by the apparatus described herein areinsufficient to damage the bacteria in such a way that the cells areforced to activate their biochemical pathways to repair the cell wall.Chemical stress can be used alone to induce damage to the cell, oralternatively can be used in combination with mechanical stress. Somenon-limiting examples of chemical stressors include, e.g., acidic pH (pH2-7), basic pH (pH 7-10), oxidative stress (e.g., exposure to hydrogenperoxide), enzymatic stress (e.g., lysostaphin, endolysins, lysozyme),osmotic stress (e.g., high or low ionic salt to add osmotic pressure tothe cells) or porins (e.g., gramicidin). One of skill in the art caneasily optimize the amount and type of chemical stressor that permitsnon-lethal damage to the immobilized bacteria, yet substantiallyrestricts entry of a reporter agent that stains dead or dying cells(e.g., fluorescent dye) in the absence of an antibiotic.

In one embodiment, the agent that causes chemical stress to bacteria islysostaphin, which is a glycylglycine endopeptidase that specificallycleaves the pentaglycine cross bridges found in the staphylococcalpeptidoglycan. At high enough concentrations, lysostaphin kills S.aureus within minutes ([MIC₉₀] 0.001 to 0.064 μg/mL) (Wu, J. A., et al.,Antimicrob Agents Chemother (2003) 47(11):3407-14). One of skill in theart can easily tailor the dose of lysostaphin necessary to chemicallydamage the cell wall to a level appropriate for use with the methods anddevices described herein (e.g., non-lethal, repairable damage).Lysostaphin may be introduced transiently or continually to effect thechanges required.

An alternate chemical damage mechanism is to introduce low levels ofbacteriophage endolysins, which specifically damage the cell walls ofStaphylococci (Donovan, D. M., et al., FEMS Microbiol Lett (2006)265(1):133-9; O'Flaherty, S., et al., J Bacteriol (2005)187(20):7161-4). The endolysin strategy is likely to be more broadlyapplicable across Staphylococci strains as compared to lysostaphin,since they have multiple putative antimicrobial activities (e.g., thephi11 endolysin is a peptidoglycan hydrolase that exhibits bothendopeptidase and amidase activities) (Donovan, D. M., et al., FEMSMicrobiol Lett (2006) 265(1):133-9). The endolysins, when purified andexposed to bacteria externally, can cause exolysis (“lysis fromwithout”). One of skill in the art can prepare a purified endolysin bye.g., amplifying an endolysin gene by PCR, cloning it into e.g., a pETvector (Novagen), over-expressing the gene in E. coli, and purifying theendolysins using a nickel column (Donovan, D. M., et al., FEMS MicrobiolLett (2006) 265(1):133-9; Sass, P. and G. Bierbaum Appl EnvironMicrobiol (2007) 73(1):347-52).

Antibiotics

Essentially any antibiotic can be used with the methods and devicesdescribed herein. Particularly relevant antibiotics include those thatare used in the clinic, however it is also contemplated herein that acandidate antibiotic can also be tested for efficacy in this manner.Examples of the different classes of antibiotics useful with the methodsand devices described herein include, but are not limited to, betalactam antibiotics, beta lactamase inhibitors, aminoglycosides andaminocyclitols, quinolones, tetracyclines, macrolides, and lincosamides,as well as glycopeptides, lipopeptides and polypeptides, sulfonamidesand trimethoprim, chloramphenicol, isoniazid, nitroimidazoles,rifampicins, nitrofurans, methenamine, and mupirocin, all of which canbe used in conjunction with the methods described herein.

In one embodiment, the antibiotic is a cell wall biosynthesis inhibitor.An exemplary family of antibiotics that inhibit cell wall biosynthesisinclude the beta lactam antibiotics (e.g., penicillin derivatives,cephalosporins, monobactams, carbapenems, and β-lactamase inhibitors).Some non-limiting examples of cell wall biosynthesis inhibitors includepenicillin, ampicillin, benzathine penicillin, benzylpenicillin(penicillin G), phenoxymethylpenicillin (penicillin V), procainepenicillin, oxacillin, methicillin, nafcillin, cloxacillin,dicloxacillin, flucloxacillin, temocillin, amoxycillin, co-amoxiclav(amoxicillin+clavulanic acid), azlocillin, carbenicillin, ticarcillin,mezlocillin, piperacillin, aztreonam, bacitracin, cephalosporin,cephalexin, cefadroxil, cefalexin, cefprozil, cefdinir, cefdiel,cefditoren, cefoperazone, cefobid, cefotaxime, cefpodoxime, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobiprole,cephalothin, cefazolin, cefaclor, cefuroxime, cefamandole, cefotetan,cefoxitin, ceftriaxone, cefotaxime, cefpodoxime, ceftazidime,carbapenem, imipenem (with cilastatin), meropenem, ertapenem, faropenem,doripenem, aztreonam, clavulanic acid, tazobactam, sulbactam,vancomycin, teicoplanin, loracarbef, and ramoplanin.

Antibiotic—Mechanism of Action

In one embodiment, it is preferred that the antibiotic used has amechanism of action that inhibits cell wall synthesis. These antibioticsare preferred because repair of the cell wall in the presence of a cellwall synthesis inhibitor is a direct measure of antibiotic resistance,since the mechanism of antibiotic action inhibits repair necessitatedthe stressor-induced damage. The methods and devices described hereinproduce cellular damage to the cell wall of a bacterium, permittingfluorescent dye to be taken up by damaged cells. The damaged cellsactivate metabolic synthesis of the cell wall in order to repair thestressor-induced damage. Therefore, susceptibility to an antibiotic thatprevents the re-synthesis of the cell wall (such as those describedabove) can be readily monitored by e.g., assessing the intensity offluorescence of a dye taken up by the damaged cells or extruded by cellsundergoing repair.

It is also contemplated herein that antibiotics having another mechanismof action can be tested using the methods described herein, even if theeffect on cell wall repair is indirect. Often changes in cellularprocesses are reflected by the state of the cell wall, which permits oneto use the methods described herein even if the antibiotic is not a cellwall synthesis inhibitor. It is well within the abilities of one ofskill in the art to adapt the methods described herein for cell wallsynthesis inhibitors, such that the methods can be used with antibioticshaving another mechanism of action. For example, antibiotics thatinhibit protein synthesis can be tested by inducing damage to the cell(e.g., by physical or chemical stress) that necessitates proteinsynthesis of e.g., peptidoglycan to repair the cell wall. A cell that isable to continue protein synthesis in the presence of the antibiotic andthus repair the cell wall is determined to be resistant to theantibiotic, while cells that cannot repair the cell wall are consideredto be susceptible to the antibiotic.

It is also contemplated herein that a physical stress as describedherein can also damage other cellular functions, whose repair can bemonitored in the presence of an antibiotic that targets that function.

Antibiotics with a different mechanism of action (e.g., DNA synthesisinhibitors, protein synthesis inhibitors etc) are also contemplated foruse and the methods described herein for cell wall synthesis inhibitorscan be generalized for use with these antibiotics. In general, thestressor-induced damage is matched with the mechanism of action of theantibiotic; that is the stressor will induce a metabolic repair pathwayto be turned on, permitting one to measure the recovery of the cell inthe presence of an antibiotic that aims to inhibit the induced metabolicpathway. For example, to test susceptibility to an antibiotic thatinhibits DNA synthesis, one would induce DNA damage by e.g., UVradiation thus activating the cell to repair DNA and then monitor DNAsynthesis rates by e.g., labeled-thymidine incorporation in the presenceof a DNA synthesis inhibitor. If there is a decrease in the level ofthymidine incorporation (e.g., the cell is unable to repair itself) inthe presence of an antibiotic, the cell is susceptible to theantibiotic. Alternatively, if there is no change in the level ofthymidine incorporation among cells in the presence and absence of theantibiotic (e.g., the cell is capable of repair), the cell is determinedto be resistant to the antibiotic.

Screening Candidate Antibiotics

The methods and devices described herein can be used to screen candidateantibiotics for efficacy against a bacterial sample, a bacterial strainor a mix of bacterial strains. In one embodiment, the device describedherein comprises a plurality of channels to which bacterial cells areimmobilized. In such a multi-channel system, one can screen a pluralityof candidate antibiotics (e.g., one antibiotic per channel) or screenone antibiotic against a plurality of bacterial strains (e.g., onebacterial strain per channel). One can also use the methods and devicesdescribed herein to test a range of doses for an antibiotic or candidateantibiotic, determine efficacy of an antibiotic, and/or to determine aminimum inhibitory concentration for an antibiotic or candidateantibiotic.

Determining Minimum Inhibitory Concentration (MIC)

As used herein, the term “minimum inhibitory concentration” refers tothe lowest concentration of an antibiotic that will inhibit the visiblegrowth of a microorganism. In the context of the present invention, theterm “minimum inhibitory concentration” also encompasses the lowestconcentration of an antibiotic that effects cell death or inhibits cellwall repair using the methods and devices described herein. In oneembodiment, the methods and devices described herein permit thedetermination of a minimum inhibitory concentration for an antibiotic orcandidate antibiotic against a bacterial strain. In one embodiment, theminimum inhibitory concentration of an antibiotic can be determined bymeasuring a modulation in the response of the bacterial cells (e.g.,uptake or extrusion of a reporter stain, change in morphology, change inmetabolism, etc) in a channel exposed to an antibiotic compared to thesame bacterial cells in a channel not exposed to the antibiotic or todifferent concentrations of the same antibiotic.

The minimum inhibitory concentration is a clinically relevant valueindicating the minimum effective dose of an antibiotic to beadministered to a subject to induce bacterial cell death and/or reduceat least one symptom of the bacterial-mediated disease. Clinically, theminimum inhibitory concentrations are used not only to determine theamount of antibiotic that a subject will receive but also to determinethe preferred antibiotic to be used. A minimum inhibitory concentrationcan also be determined for a candidate antibiotic to permit e.g.,efficacy determination and dosing information for clinical trials.

One can easily test a range of antibiotic concentrations using e.g., the4-channel device described herein in the Example section or anotherdevice comprising a plurality of channels as described herein todetermine a dose response relationship for the antibiotic.

Devices

In some embodiments, a device as described herein is contemplated tohave a plurality of channels (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 15, 20, 24, 25, 30, 36, 40, 48, 50, 55, 60, 70, 80, 90, 100, ormore) with each channel corresponding to a different antibiotic and/or adifferent bacterial sample. In addition, each channel can correspond toa different dose or concentration of a particular antibiotic testedagainst an immobilized bacterial strain. In one embodiment, theplurality of channels are arranged in parallel. The channels can beformed using e.g., silicone or silicone rubber as described, forexample, herein in the Examples section (FIGS. 5, 6, and 7). In oneembodiment, the channels are disposable, but it is also contemplatedherein that the channels are re-used with the methods and devices usedherein. The device described herein is designed for use with a solidsupport to which a bacterium is immobilized. It is preferred that thesolid support is disposable to prevent cross-contamination betweenbacterial samples. The solid supports are entirely removable from thedevice but are used with the assay system described e.g., in the methodsdescribed herein. In one embodiment, the device comprises a microscopewith an optical stage that can be controlled in the X, Y and Z planes.

In one embodiment, a sample derived from a patient can be testedconcurrently with multiple antibiotics in a plurality of channels. Whileit can be manually detected (e.g., using a microscope), in oneembodiment the device detects e.g., fluorescence emission and theintensity of a signal using an agent as described herein and displaysthe location (e.g., channel) matching the antibiotic for the user. Ifseveral antibiotics produce a detectable signal, the antibiotic with thehighest intensity of signal is then administered to the patient by askilled clinician.

In some embodiments, the device further comprises immobilized bacteria.Such a device is particularly useful for screening candidate antibioticmolecules. It is contemplated herein that such a device comprises aplurality of channels to permit high-throughput screening of a e.g.,small molecule library. The device comprises a detector that detectsfluorescence intensity concurrently from the plurality of channels.Fluorescence data from the detector is then stored and/or the computerdetermines the location of a fluorescence signal on e.g., a chipcomprising the plurality of channels. The computer then displays thelocation of any molecules that have antibiotic efficacy.

In one embodiment, the device described herein comprises at least onepump to control the flow velocity or flow rate of a medium through achannel lined with bacterial cells. In one embodiment, a single pump isused to achieve a flow velocity consistent across a plurality ofchannels. This has the added advantage of maintaining substantiallysimilar assay conditions for the antibiotic testing channel(s) comparedto one or more control channels that lack antibiotic, and permitting thedetected levels of the reporter stain in the testing channel(s) to benormalized to background levels in the control channel(s). In oneembodiment, the flow velocity in each channel of a plurality of channelsin the device can be controlled by a plurality of pumps (e.g., 1 pumpper channel, 1 pump/2 channels, 1 pump/5 channels, 1 pump/10 channels).

In one embodiment of the methods and devices described herein, themethod is completely automated. The device can be automated (e.g., usingsoftware) such that the total fluorescence in a location of each channelis scanned, measured and/or delivered to a computer or other datastorage device. Each channel can be scanned for e.g., fluorescencesimultaneously with other channels or in a sequential manner where onechannel is scanned after another. The channels are scanned iterativelyduring the assay process, however the location within the channel (e.g.,optical view) for each iterative measurement will be substantiallyidentical to prevent artifactual measurements of reporter stainintensity. The timing (e.g., cycle) between each iterative measurementcan be chosen by one of skill in the art. For example, the intensity ofthe reporter stain (e.g., fluorescence) in a channel can be measuredevery 30 sec, every minute, every 2 min, every 3 min, every 4 min, every5 min, every 10 min, every 15 min, every 20 min, every 30 minutes ormore over a length of time desired by one of skill in the art. Inanother embodiment, the device described herein comprises a handhelddevice that is entirely portable. While not necessary for the methodsdescribed herein, in one embodiment the device further comprises amicroscope. Alternatively, in another embodiment, the device does notcomprise a microscope.

Systems

Embodiments of the invention also provide for systems (and computerreadable media for causing computer systems) to perform a method fordetermining whether a bacterium is susceptible to an antibiotic bymeasuring e.g., fluorescence emission.

Embodiments of the invention have been described through functionalmodules, which are defined by computer executable instructions recordedon computer readable media and which cause a computer to perform methodsteps when executed. The modules have been segregated by function forthe sake of clarity. However, it should be understood that themodules/systems need not correspond to discreet blocks of code and thedescribed functions can be carried out by the execution of various codeportions stored on various media and executed at various times.Furthermore, it should be appreciated that the modules may perform otherfunctions, thus the modules are not limited to having any particularfunctions or set of functions.

The computer readable media can be any available tangible media that canbe accessed by a computer. Computer readable media includes volatile andnonvolatile, removable and non-removable tangible media implemented inany method or technology for storage of information such as computerreadable instructions, data structures, program modules or other data.Computer readable media includes, but is not limited to, RAM (randomaccess memory), ROM (read only memory), EPROM (eraseable programmableread only memory), EEPROM (electrically eraseable programmable read onlymemory), flash memory or other memory technology, CD-ROM (compact discread only memory), DVDs (digital versatile disks) or other opticalstorage media, magnetic cassettes, magnetic tape, magnetic disk storageor other magnetic storage media, other types of volatile and nonvolatilememory, and any other tangible medium which can be used to store thedesired information and which can accessed by a computer including andany suitable combination of the foregoing.

Computer-readable data embodied on one or more computer-readable mediamay define instructions, for example, as part of one or more programs,that, as a result of being executed by a computer, instruct the computerto perform one or more of the functions described herein, and/or variousembodiments, variations and combinations thereof. Such instructions maybe written in any of a plurality of programming languages, for example,Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic,COBOL assembly language, and the like, or any of a variety ofcombinations thereof. The computer-readable media on which suchinstructions are embodied may reside on one or more of the components ofeither of a system, or a computer readable medium described herein, maybe distributed across one or more of such components, and may be intransition there between.

The computer-readable media may be transportable such that theinstructions stored thereon can be loaded onto any computer resource toimplement the aspects of the present invention discussed herein. Inaddition, it should be appreciated that the instructions stored on thecomputer-readable medium, described above, are not limited toinstructions embodied as part of an application program running on ahost computer. Rather, the instructions may be embodied as any type ofcomputer code (e.g., software or microcode) that can be employed toprogram a computer to implement aspects of the present invention. Thecomputer executable instructions may be written in a suitable computerlanguage or combination of several languages. Basic computationalbiology methods are known to those of ordinary skill in the art and aredescribed in, for example, Setubal and Meidanis et al., Introduction toComputational Biology Methods (PWS Publishing Company, Boston, 1997);Salzberg, Searles, Kasif, (Ed.), Computational Methods in MolecularBiology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler,Bioinformatics Basics: Application in Biological Science and Medicine(CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: APractical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc.,2nd ed., 2001).

The functional modules of certain embodiments of the invention includeat minimum a determination system, a storage device, a comparison moduleand a display module. The functional modules can be executed on one, ormultiple, computers, or by using one, or multiple, computer networks.The determination system has computer executable instructions to providee.g., fluorescence information in computer readable form.

The determination system, #40, can comprise any system for detecting asignal from an agent comprising a reporter moiety. Such systems caninclude, flow cytometry systems, fluorescence assisted cell sortingsystems, fluorescence microscopy systems (e.g., fluorescence microscopy,confocal microscopy), and other systems for measuring fluorescenceemission. It is contemplated herein that a detector from a microfluidicssystem can be used as the determination system.

The information determined in the determination system can be read bythe storage device #30. As used herein the “storage device” is intendedto include any suitable computing or processing apparatus or otherdevice configured or adapted for storing data or information. Examplesof electronic apparatus suitable for use with the present inventioninclude stand-alone computing apparatus, data telecommunicationsnetworks, including local area networks (LAN), wide area networks (WAN),Internet, Intranet, and Extranet, and local and distributed computerprocessing systems. Storage devices also include, but are not limitedto: magnetic storage media, such as floppy discs, hard disc storagemedia, magnetic tape, optical storage media such as CD-ROM, DVD,electronic storage media such as RAM, ROM, EPROM, EEPROM and the like,general hard disks and hybrids of these categories such asmagnetic/optical storage media. The storage device is adapted orconfigured for having recorded thereon sequence information orexpression level information. Such information may be provided indigital form that can be transmitted and read electronically, e.g., viathe Internet, on diskette, via USB (universal serial bus) or via anyother suitable mode of communication.

As used herein, “stored” refers to a process for encoding information onthe storage device. Those skilled in the art can readily adopt any ofthe presently known methods for recording information on known media togenerate manufactures comprising the sequence information or expressionlevel information.

In one embodiment the reference data stored in the storage device to beread by the comparison module is fluorescence emission data obtainedfrom a stressed bacterium in the absence of antibiotic.

The “comparison module” #80, 90 can use a variety of available softwareprograms and formats for the comparison operative to comparefluorescence data determined in the determination system to referencesamples and/or stored reference data. In one embodiment, the comparisonmodule is configured to use pattern recognition techniques to comparesequence information from one or more entries to one or more referencedata patterns. The comparison module may be configured using existingcommercially-available or freely-available software for comparingpatterns, and may be optimized for particular data comparisons that areconducted. The comparison module provides computer readable informationrelated to the antibiotic susceptibility that can include, for example,detection of fluorescence, fluorescence intensity etc.

The comparison module, or any other module of the invention, may includean operating system (e.g., UNIX) on which runs a relational databasemanagement system, a World Wide Web application, and a World Wide Webserver. World Wide Web application includes the executable codenecessary for generation of database language statements (e.g.,Structured Query Language (SQL) statements). Generally, the executableswill include embedded SQL statements. In addition, the World Wide Webapplication may include a configuration file which contains pointers andaddresses to the various software entities that comprise the server aswell as the various external and internal databases which must beaccessed to service user requests. The Configuration file also directsrequests for server resources to the appropriate hardware—as may benecessary should the server be distributed over two or more separatecomputers. In one embodiment, the World Wide Web server supports aTCP/IP protocol. Local networks such as this are sometimes referred toas “Intranets.” An advantage of such Intranets is that they allow easycommunication with public domain databases residing on the World WideWeb (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in aparticular preferred embodiment of the present invention, users candirectly access data (via Hypertext links for example) residing onInternet databases using a HTML interface provided by Web browsers andWeb servers.

The comparison module provides a computer readable comparison resultthat can be processed in computer readable form by predefined criteria,or criteria defined by a user, to provide a content based in part on thecomparison result that may be stored and output as requested by a userusing a display module #110.

The content based on the comparison result, #100, may be a fluorescenceintensity profile showing comparative sensitivity among a panel ofantibiotics #140. In one embodiment, the content based on the comparisonresult is a signal indicative of the susceptibility of a bacterium to aparticular antibiotic #140.

In one embodiment of the invention, the content based on the comparisonresult is displayed on a computer monitor #120. In one embodiment of theinvention, the content based on the comparison result is displayedthrough printable media #130. The display module can be any suitabledevice configured to receive from a computer and display computerreadable information to a user. Non-limiting examples include, forexample, general-purpose computers such as those based on IntelPENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC,Hewlett-Packard PA-RISC processors, any of a variety of processorsavailable from Advanced Micro Devices (AMD) of Sunnyvale, Calif., or anyother type of processor, visual display devices such as flat paneldisplays, cathode ray tubes and the like, as well as computer printersof various types.

In one embodiment, a World Wide Web browser is used for providing a userinterface for display of the content based on the comparison result. Itshould be understood that other modules of the invention can be adaptedto have a web browser interface. Through the Web browser, a user mayconstruct requests for retrieving data from the comparison module. Thus,the user will typically point and click to user interface elements suchas buttons, pull down menus, scroll bars and the like conventionallyemployed in graphical user interfaces.

The present invention therefore provides for systems (and computerreadable media for causing computer systems) to perform methods fordetermining whether a bacterium is susceptible to a particularantibiotic.

Systems and computer readable media described herein are merelyillustrative embodiments of the invention for performing methods ofdetermining whether a bacterium is susceptible to an antibiotic, and isnot intended to limit the scope of the invention. Variations of thesystems and computer readable media described herein are possible andare intended to fall within the scope of the invention.

The modules of the machine, or used in the computer readable medium, mayassume numerous configurations. For example, function may be provided ona single machine or distributed over multiple machines.

Advantages of the Methods Described Herein

The current methodologies for measuring antimicrobial susceptibilityinclude dilution testing, disk diffusion, Etest, latex agglutination,specialized CHROMagars, mechanized or semi-automated systems based ondilution testing, and PCR. These current standard methods for detectingantibiotic susceptibility are based on the ability of the bacteria toproliferate in the presence of antibiotics, and thus these techniquesare time-consuming, costly, and insensitive, particularly for evaluationof slow-growing organisms. To develop a truly rapid susceptibility test,one must circumvent the need for growth.

Described herein is a novel method for rapid antibiotic susceptibilitydetection in e.g., less than an hour. The methods and devices describedherein have the potential to revolutionize clinical practice forbacterial infections by enabling clinicians to prescribe appropriateantibiotic therapy much sooner than is possible with current methods.The methods and devices can also be multiplexed (multiple antibioticsand/or multiple organisms) and can be integrated with current bacterialidentification methodologies.

The methods and devices described herein can also be used to complementexisting rapid tests based on molecular diagnostics (e.g. PCR) becausethey would provide a low-cost rapid method that delivers clinicallyactionable information (susceptibility profiles). While genetic testsprovide precise information for epidemiological studies, the highreagent costs, relatively high operator skills required, and limitedclinical utility continue to limit widespread routine use. Furthermore,the molecular diagnostics suffer from a high number of false positivesand unacceptable performance in non-sterile specimens due to theco-presence of methicillin-sensitive S. aureus and methicillin-resistantcoagulase-negative staphylococci (von Eiff, C., et al., J AntimicrobChemother (2008) 61(6):1277-80; E Becker, K., et al., J Clin Microbiol(2006) 44(1):229-31). This limitation has been overcome in some assaysby linking detection of the mecA gene with detection of a neighboringorfX gene, however flanking regions can be heterogeneous and lead tofalse negatives (Huletsky, A., et al., J Clin Microbiol (2004) 42(5):1875-84). Since the methods described herein can be automated and arebased on phenotypic changes, the methods are useful for routine clinicaldiagnostic and providing clinicians with the information they need totreat their patients, namely what antibiotic to use and at what dosage.

Rapid methods, such as the methods described herein, has the followingadvantages as compared to traditional methods: (1) informs physiciansand influences their treatment decisions sooner (Matsen, J. M., DiagnMicrobiol Infect Dis (1985) 3(6 Suppl):73S-78S; Vincent, P., Presse Med(1985) 14(32):1697-700), (2) reduces mortality rates (Doern, G. V., etal., J Clin Microbiol (1994) 32(7):1757-62), (3) significantly reducesthe number of laboratory studies, imaging procedures, days ofintubation, and days spent in the intensive care unit (Doern, G. V., etal., J Clin Microbiol (1994) 32(7):1757-62), and (4) reduces the overallcosts of hospitalization (Doern, G. V., et al., J Clin Microbiol (1994)32(7):1757-62; Barenfanger, J., et al., J Clin Microbiol (1999)37(5):1415-8). Moreover, as bacterial populations become more resistantto available antibiotics, it becomes more critical that physicians moveto targeted narrow-spectrum antibiotic therapy. This can only beaccomplished with the assistance of new antibiotic susceptibilitymethodologies such as the methods described herein that provide resultsin a fraction of the time of current protocols.

Traditional Tests for Antibiotic Susceptibility

The traditional tests for antibiotic susceptibility and the limitationsof these tests are described herein below.

Traditionally, antibiotic susceptibility was determined either by adilution or disk diffusion technique (Henry, J. B., ed. ClinicalDiagnosis and Management by Laboratory Methods. 20th ed. 2001, Saunders:Philadelphia). In the dilution test, the microorganism is inoculatedinto a series of tubes or wells containing a range of concentrations ofthe antibiotic. The lowest inhibitory antibiotic concentration is termedthe minimum inhibitory concentration (MIC). In the disk diffusion test,a paper disk containing a specified amount of antibiotic is applied toan agar surface that has been freshly inoculated with a lawn ofmicroorganisms. The antimicrobial diffuses from the disk, resulting in azone of inhibition at which a critical concentration of the antibioticin the medium inhibits growth at a particular point in time (typically18-24 hrs after inoculation). The zone diameter is measured and comparedto standards to define susceptibility and resistance. The zone diameteris inversely related to the MIC, i.e. the larger zones indicate lowerMICs.

The Etest is based on the diffusion of a continuous concentrationgradient of an antibiotic from a plastic strip into an agar medium. Theplastic strip has a predefined concentration of dried drug on one sideand a MIC scale on the other which allows a read out of the zone ofinhibition and corresponding MIC after incubating the Etest strip afreshly inoculated microbial lawn under the appropriate conditions.Although the Etest's ease of use makes it a preferred option overtraditional diffusion or dilution testing, the cost is prohibitive formost laboratories to use on a routine basis for primary screening.

Specifically for identifying methicillin resistance in Staphylococcusaureus strains, Oxoid offers a rapid latex agglutination test for PBP2awherein latex particles sensitized with a monoclonal antibody againstPBP2a will react with MRSA to cause macroscopic agglutination. The kitincludes two extraction reagents, and the agglutination reaction isperformed on heated extracts of bacterial colonies. MRSA colonies can bedetected within 15 min; however latex agglutination kits tend to be lesssensitive than other susceptibility methods. For example, when comparingthis kit to the oxacillin screen agar method using isolated coloniesfrom blood cultures positive for S. aureus (n=70), the direct PBP2a testwas only 18% sensitive.

According to the results from proficiency testing programs of theCollege of American Pathologists, most labs in the U.S. now usemicrodilution based semi-automated systems (Henry, J. B., ed. ClinicalDiagnosis and Management by Laboratory Methods. 20th ed. 2001, Saunders:Philadelphia). The shift away from the more traditional methods is aresponse to the efficiency gains from replicate inoculations of combinedidentification and susceptibility systems and the need for datamanagement systems. Two popular systems are the VITEK (BioMerieux) andMicroscan WalkAway (Dade Behring). In the VITEK system, antimicrobialsare contained in miniature wells on a plastic card. The cards areincubated in the associated reader/incubator instrument, and the wellsare monitored via optical density. Although bacterial identification canbe obtained within 4 hours, the mean time of incubation forantimicrobial susceptibility testing is approximately 8 hours (Eigner,U., et al., J Clin Microbiol (2005) 43(8): p. 3829-34). The MicroscanWalkaway conducts endpoint photometric measurements, for which bacterialidentification is available approximately within 3-5 hours, butantibacterial susceptibility results are available no sooner than 9hours (Henry, J. B., ed. Clinical Diagnosis and Management by LaboratoryMethods. 20th ed. 2001, Saunders: Philadelphia; Sellenriek, P., et al(2005) 105th General Meeting of the American Society for MicrobiologyAtlanta, Ga.). However a recent evaluation of the Microscan forsusceptibility testing of gram positive organisms found it took anaverage of 23.3 hours before susceptibility results were available(McCarter, Y. S., et al., 105th General Meeting of the American Societyfor Microbiology (2005) Atlanta, Ga.). One advantage of the Microscan isthat the results are also interpretable by eye if there is a problemwith the instrument, which is an appealing feature to lab directors.

In recent years, several targeted methods have emerged in themarketplace to address the need for rapid detection of specificpathogens. In the case of MRSA, two noteworthy offerings are availablefrom Becton Dickenson; CHROMagar MRSA and GeneOhm StaphSR assay. TheCHROMagar MRSA is a selective and differential medium for thequalitative direct detection of nasal colonization by methicillinresistant S. aureus (MRSA) to aid in the prevention and control of MRSAinfections in healthcare settings. The test is performed on anteriornares swabs to screen for MRSA colonization but is not intended todiagnose MRSA infection or provide information to guide treatment, suchas MIC or susceptibility profiles. The results from a CHROMagar plateare available within 24-48 hrs, but occasional strains ofcoagulase-negative staphylococci or corynebacteria may grow onCHROMagar. Thus, a confirmatory coagulase test is necessary for MRSAconfirmation. The GeneOhm StaphSR assay is a PCR based assay thatrapidly provides simultaneous identification of methicillin-susceptibleS. aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA)and was recently cleared by the FDA for the direct detection of MRSAfrom patients with positive blood cultures and for individuals positivefor MRSA nasal colonization. The GeneOhm assay provides targetedinformation (SA identification and mecA detection) within a few hours,but again does not provide phenotypic information (MIC or susceptibilityprofiles). These methods are useful for screening, but do not providethe critical information that a physician needs for treatment, namelywhich antibiotic to prescribe to the patient. Furthermore, although thetechniques are rapid, they are also significantly more expensive thanthe more traditional methods. The combination of incomplete informationand increased cost limit the usefulness of these tests in routine use.

The more traditional methods, such as dilution, disk diffusion, or Etesttechniques, are accurate and inexpensive, but are lengthy and requiresignificant operator time. The accuracy of these methods depends onstrict adherence to standard methods, including the medium used,inoculation size, and incubation conditions (Henry, J. B., ed. ClinicalDiagnosis and Management by Laboratory Methods. 20th ed. 2001, Saunders:Philadelphia). The automated susceptibility systems provide decreases inturn-around time and operator time, but can add significant cost to thetests. Moreover these semi-automated systems do not provide informationin time to influence initial treatment decisions. PCR and CHROMagars canbe useful in outbreaks, but are not appropriate for broad spectrumdiagnosis due to limited information and high cost.

Flow Cytometry Methods

As early as 1982, researchers began studying changes in bacteriaincubated with various active agents via flow cytometry (Steen, H. B.,et al., Cytometry (1982) 2(4):249-57). The early studies focused onmorphology, cell cycle kinetics, DNA replication, and bacterialmetabolism (Steen, H. B., et al., supra; Boye, E. and A. Lobner-OlesenRes Microbiol (1991) 142(2-3):131-5; Fouchet, P., et al., Biol Cell(1993) 78(1-2): 95-109; Martinez, O. V., et al., Cytometry (1982)3(2):129-33). The general format of the studies includes treatment ofthe cells with an active agent, staining the cells with fluorescentdyes, and then monitoring the response of the active agents over timevia flow cytometry. Several authors demonstrated monitoring of bacteriaviability and vitality using flow cytometry, including various strainsof Escherichia coli (Mason, D. J., et al., J Appl Bacteriol (1995)78(3):309-15; Jepras, R. I., et al., Appl Environ Microbiol (1995)61(7):2696-2701; Kaprelyants, A. S. and D. B. Kell Journal of AppliedBacteriology (1992) 72:410-422), Salmonella typhimurium (Mason, D. J.,et al., (1995), supra), Staphylococcus spp. (Mason, D. J., et al.,(1995), supra; Jepras, R. I., et al., (1995), supra, Lloyd, D. and A. J.Hayes, FEMS Microbiol Lett (1995) 133:1-7; Langsrud, S. and G. SundheimJ Appl Bacteriol (1996) 81(4):411-8), Bacillus subtilis (Diaper, J. P.,et al., Appl Microbiol Biotechnol (1992) 38(2):268-72), Pseudomonas spp.(Jepras, R. I., et al., Appl Environ Microbiol (1995) 61(7): 2696-2701;Langsrud, S, and G. Sundheim J Appl Bacteriol (1996) 81(4): 411-8) andMicrococcus luteus (Kaprelyants, A. S. and D. B. Kell, Journal ofApplied Bacteriology (1992) 72:410-422).

Subsequently, a number of groups demonstrated rapid detection ofantibiotic susceptibility or resistance via flow cytometric measurementsof cultured bacteria (Martinez, O. V., et al., Cytometry (1982)3(2):129-33; Suller, M. T., et al., J Antimicrob Chemother (1997)40(1):77-83; Durodie, J., K. et al., Cytometry (1995) 21(4):374-7;Ordonez, J. V. and N. M. Wehman, Cytometry (1993) 14(7): 811-8; Pore, R.S. J Antimicrob Chemother (1994) 34(5):613-27; Roth, B. L., et al., ApplEnviron Microbiol (1997) 63(6):2421-31; Mason, D. J., et al. J Microsc,(1994) 176(Pt 1):8-16; Mason, D. J. and V. A. Gant, J AntimicrobChemother, (1995) 36(2): 441-3; Gant, V. A., et al., J Med Microbiol,(1993) 39(2): 147-54). and directly from clinical specimens (Cohen, C.Y. and E. Sahar, J Clin Microbiol (1989) 27(6):1250-6). The bacteriastudied include both gram negative and gram positive organisms;Escherichia coli (Martinez, O. V., et al., Cytometry (1982) 3(2):129-33;Durodie, J., K. et al., Cytometr (1995) 21(4):374-7; Roth, B. L., etal., Appl Environ Microbiol (1997) 63(6):2421-31; Mason, D. J., et al.,J Microsc (1994) 176(Pt 1):8-16), Bacillus cereus (Roth, B. L., et al.,Appl Environ Microbiol (1997) 63(6):2421-31), S. aureus (Suller, M. T.,et al., J Antimicrob Chemother (1997) 40(1):77-83; Ordonez, J. V. and N.M. Wehman, Cytometry (1993) 14(7):811-8; Roth, B. L., et al., ApplEnviron Microbiol (1997) 63(6):2421-31; 44; Cohen, C. Y. and E. Sahar,(1989), supra), Staphylococcus epidermidis (Cohen, C. Y. and E. Sahar,(1989), supra), Streptococcus pyogenes (Cohen, C. Y. and E. Sahar,(1989), supra), Klebsiella pneumoniae (Cohen, C. Y. and E. Sahar,(1989), supra), Pseudomonas aeruginosa (Cohen, C. Y. and E. Sahar,(1989), supra), P. stutzeri (Cohen, C. Y. and E. Sahar (1989), supra),Proteus mirabilis (Cohen, C. Y. and E. Sahar (1989), supra), andEnterobacter spp. (Cohen, C. Y. and E. Sahar, (1989), supra).

Several notable results come from this body of work. First, researcherswere able to accurately determine sensitivity to various antibiotics,including ampicillin (Roth, B. L., et al., Appl Environ Microbiol (1997)63(6):2421-31; Mason, D. J., et al., J Microsc (1994)176(Pt 1):8-16),amoxicillin (Roth, B. L., et al., Appl Environ Microbiol (1997)63(6):2421-31), penicillin G (Suller, M. T., et al., J AntimicrobChemother (1997) 40(1):77-83, 41, Roth, B. L., et al., (1997), supra],vancomycin (Suller, M. T., et al., J Antimicrob Chemother (1997)40(1):77-83; Roth, B. L., et al., (1997), supra), gentamicin (Mason, D.J., et al., J Microsc (1994)176(Pt 1):8-16), ciprofloxacin (Durodie, J.,K. et al., Cytometr (1995) 21(4):374-7); Mason, D. J., et al., J Microsc(1994)176(Pt 1):8-16), methicillin (Suller, M. T., et al., J AntimicrobChemother (1997) 40(1):77-83), amoxicillin (Durodie, J., K. et al.,Cytometr (1995) 21(4):374-7), mecillinam (Durodie, J., K. et al.,Cytometr (1995) 21(4):374-7), chloramphenicol (Durodie, J., K. et al.,Cytometr (1995) 21(4):374-7), trimethoprim (Durodie, J., K. et al.,Cytometr (1995) 21(4):374-7), sodium cefazolin (Martinez, O. V., et al.,Cytometry (1982) 3(2):129-33), moxalactam (Martinez, O. V., et al.,Cytometry (1982) 3(2):129-33), cefamandole lithium (Martinez, O. V., etal., Cytometry (1982) 3(2):129-33), oxacillin (Ordonez, J. V. and N. M.Wehman, Cytometry (1993) 14(7):811-8), and amikacin (Cohen, C. Y. and E.Sahar, J Clin Microbiol (1989) 27(6):1250-6). The antibiotics spannedvarious antibiotic classes (β-lactams, cephalosporins, fluoroquinolones,glycopeptides, aminoglycosides, etc.), which have distinct mechanisms ofaction, ranging from inhibiting cell wall biosynthesis to interferingwith protein synthesis or DNA replication. The susceptibility was wellcorrelated with standard disk diffusion or dilution methods.

Second, antibiotic susceptibility was determined very rapidly (30min-2.5 hrs, depending on the bacterial species under study and theprotocol used). The rapid results were achieved because the techniquedoes not require time for population growth, but instead interrogatesthe direct effect of antibiotics on cellular viability for a smallnumber of bacteria.

Third, a consensus of the utility of various fluorescent dyes emerged.Although several dyes were used to demonstrate rapid antibioticsusceptibility via flow cytometry, including Rhodamine 123 (Mason, D.J., et al., J Appl Bacteriol (1995) 78(3):309-15; Kaprelyants, A. S. andD. B. Kell, (1992), supra, Langsrud, S, and G. Sundheim J Appl Bacteriol(1996) 81(4): 411-8), Sytox green (Langsrud, S. and G. Sundheim J ApplBacteriol (1996) 81(4): 411-8; Roth, B. L., et al., (1997), supra), LIVEstain (Langsrud, S. and G. Sundheim J Appl Bacteriol (1996) 81(4):411-8), carbocyanines (e.g. 3,3′-dipentyloxacarbocyanine iodide,DiOC5(3)) (Mason, D. J., et al., (1995), supra; Ordonez, J. V. and N. M.Wehman, Cytometry (1993) 14(7):811-8), propidium iodide (Jepras, R. I.,et al., Appl Environ Microbiol (1995) 61(7): 2696-2701; Roth, B. L., etal., (1997), supra), ethidium monoazide (Jepras, R. I., et al., ApplEnviron Microbiol (1995) 61(7): 2696-2701), FITC (Durodie, J., et al.,Cytometry, (1995) 21(4):374-7), ethidium bromide (Martinez, O. V., etal., Cytometry (1982) 3(2):129-33; Jepras, R. I., et al., (1995), supra;Cohen, C. Y. and E. Sahar, (1989), supra), fluorescein esters (Jepras,R. I., et al., (1995), supra), calcafluor white (Mason, D. J., et al.,(1995), supra), and an oxonol (bis-(1,3-dibutyl-barbituricacid)trimethine oxonol, DiBAC₄(3)) (Mason, D. J., et al., (1995), supra;Suller, M. T., et al., J Antimicrob Chemother (1997) 40(1):77-83; Mason,D. J., et al., J Microsc (1994)176(Pt 1):8-16), only a couple appear tobe well-suited for a broad-based diagnostic technique. In oneembodiment, detection as described herein, is performed using flowcytometry methods. In an alternate embodiment, detection is notperformed using flow cytometry methods.

Although it has been shown that antibiotic susceptibility/resistance canbe assessed on intact bacterial cells using flow cytometry methods, thismethod still takes between 30 minutes and 2.5 hours to produce a result.The discovery that shear stress can expedite the process of detectingbacterial antibiotic susceptibility permits a result to be determinedwithin 1-30 minutes. In one embodiment, the bacteria can be testedwithin the range of 5-30 minutes inclusive; in alternate embodiments thebacteria can be tested in 5-25 minutes, 5-20 minutes, 5-10 minutes, 5-8minutes, 1-2 minutes, 1-5 minutes, 1-10 minutes, 1-15 minutes, 15-20minutes, 15-25 minutes or 15-30 minutes. In some embodiments, a resultis produced using the methods and devices described herein within 30-120minutes. It is preferred that an appropriate antibiotic

can be selected for treatment of an individual within the time frame ofa typical doctor's visit. Such a visit generally lasts about 20 minutes,so to develop a method that would enable proper targetedantibiotic-bacterial matching in less than 20 minutes wouldsignificantly reduce costs associated with healthcare professionals'time. Thus, it is contemplated herein that a sample is obtained andanalyzed within this time frame so that the doctor can appropriatelyprescribe an antibiotic that will be beneficial in treatment of theindividual.

Optimization of the Methods Described Herein

Herein are described exemplary considerations for optimizing variousparameters including fluorescent stains, growth conditions (medium,temperature etc.), flow rates and addition of chemical stressors.

As described herein, exemplary stains for use with the methods anddevices described herein are Sytox green and DiBAC₄(3). Both stains meetthe criteria of effectively staining gram positive and gram negativebacteria, are non-toxic, only stain damaged cells, can be used directlyin the growth medium, and do not require additional pretreatmentprocessing steps. Additionally, both stains are commercially availablefrom Molecular Probes. The bacteria are first bound to the glass slidesand the stain is flowed past the cells to stain them. The stainingconditions can be varied to identify the best staining parameters,including stain concentration, ranging from 0.025-0.5 μM for Sytox(Langsrud, S. and G. Sundheim, J Appl Bacteriol (1996) 81(4):411-8;Roth, B. L., et al., Appl Environ Microbiol (1997) 63(6):2421-31) and0.25-2.0 mg/L for DiBAC₄(3) (Suller, M. T., J. M. Stark, and D. Lloyd JAntimicrob Chemother (1997) 40(1):77-83; Mason, D. J., et al., J Microsc(1994) 176(Pt 1):8-16), and change in stain intensity over time toidentify the time required for staining. The utility of the stains inthe flow cell are assessed by comparing live cells grown to mid-logphase, and dead cells (both heat-killed and gramicidin-treated). Thesecontrols provide the range of signals expected and allow calibration ofthe image capture software.

The non-toxicity of the stains at the optimized concentrations can beconfirmed via standard quantitative plating techniques performed onstained and unstained bacterial suspensions. For example, to test S.aureus in mid-log phase, cultures can be inoculated with MHB+2% NaCl toa density of ˜10⁷ cfu/ml and the culture is incubated at 35° C. untilthe optical density of the culture at 650 nm reaches ˜0.35. The bacteriaare harvested by centrifugation, washed once, and then immobilized onthe glass slide surface. The Sytox stain has a low level of backgroundthat allows cells to be visualized without the addition of other dyes(Roth, B. L., et al., Appl Environ Microbiol (1997) 63(6):2421-31).However, a positive control dye can be added to the cells to assure thatan adherent population of cells has been achieved and that a lack ofsignal does not mean an absence of cells. In one embodiment it ispreferred to add as few stains to the experiment as possible so as notto disrupt the interactions with the antibiotics and thus Sytox may bepreferred for this reason.

Once the staining procedure is determined for a particular fluorescentdye, the flow parameters can be varied to ascertain the level ofmechanical stress via shear stress necessary to produce cell damage of aparticular strain of bacteria in order to detect antibioticsusceptibility.

An antibiotic (e.g., oxacillin) can be added at the minimum inhibitoryconcentration (MIC), and also 0.1×, 0.5×, 5×, and 10× MIC to optimizethe assay for a particular set of flow parameters.

The flow parameters to be used can include application of static flow atvarious velocities and application of increasing flow velocities atvarious ramping speeds. It is further contemplated herein that aconstant shear stress is applied over time or an increasing shear stressis applied over time. The flow parameters to be used can also includeintermittent flows varying between no flow and fast flows, or slowerflows and faster flows, or any combination thereof.

Additionally, the effect of various growth environments for the bacteriacan be tested. It is likely that at least minimal media is required tosustain the bacteria and allow them to repair any damage caused bymechanical stress in combination with the antibiotics. Thus, in generalthe methods described herein use standard media as the fluid (i.e.Mueller Hinton broth), but can be varied in terms of concentration,media type, and temperature of the fluid. The optimization of thebacterial environmental conditions is especially important if the cellsin the absence of antibiotics are rapidly damaged under mild shearconditions.

Bacterial Strains

Optimization of the methods described herein can be performed using twostrains of bacterial cells grown to logarithmic phase (MSSA USA300 andMRSA USA300). Initial testing with logarithmic phase bacteria was chosenbecause they are more likely to be adherent due to higher expression ofadhesins and their peptidoglycan layer is likely to be less cross-linkedand thick compared to stationary-phase cells and the cells are moremetabolically active allowing for faster response to damage.

It is further contemplated herein that optimal conditions can vary fromstrain to strain. Since different strains are often encountered in aclinical setting, this information is important for assessing theutility of the diagnostic methodology. Although it is contemplatedherein that there will be strain variability, it is anticipated that thebacteria will behave similarly enough to permit the use of a singleprotocol for testing all the strains. This expectation is based on thefact that bacterial families (e.g., staphylococci) are genetically quitesimilar to each other and thus have similar cell structures, which willbe the main component in their responsiveness to shear stress.

In terms of bacterial strain effects, two sources of variability areexpected: differences in response time and adhesion strength. Thus onewill need to quantify the differences and then adjust the protocol toaccommodate them. For example, if strain A takes 5 min to demonstratesusceptibility, but strain B takes 30 min, then the protocol willrequire at least 30 min to determine susceptibility for an unknownstrain. Because the adhesin and capsule expression will differ amongstrains, bacteria can be covalently linked to the substrate.

Another strain difference is that although most strains of S. aureuswith reduced susceptibility to methicillin produce the low-affinitypenicillin binding protein PBP2a, encoded by the mecA gene, some strainsproduce borderline methicillin-resistance due to hyperproduction of betalactamase. For these studies, a beta-lactamase inhibitor, such asclavulanic acid can be included, although clinicians often prefer toprescribe vancomycin in these cases as opposed to Augmentin (amoxicillinwith clavulanic acid). As described previously, different combinationsof antibiotics can be tested in different channels simultaneously.

Bacterial Growth Phase

The method can be widely applied after assessing and characterizing anyvariability due to the growth phase of the bacteria. First, thetechnique can be inserted into the current clinical microbiologylaboratory workflow by simply replacing the antibiotic susceptibilitytesting that follows primary culture. In this embodiment, the primaryculture grown to stationary phase seeds the susceptibility testingapparatus. Alternatively, the technique is coupled with a rapidisolation technique to detect antibiotic susceptibility of bacteriaobtained directly from a clinical sample. This latter method has greaterclinical impact on patient management by providing antibioticsusceptibility information in time for the initial treatment decision.

In addition, bacteria can be in various growth phases providing bothintra-sample and inter-sample variations. The growth phase of thebacteria influences the results in several ways. First, bacteria in lagor stationary phase causes bacteria to respond more slowly toenvironmental stressors, thus there can be differences in time toresults as compared to actively growing bacteria. Second, the adherenceto the capture agents varies due to differing expression of adhesins andcapsule production in post-exponential phase cultures. This cannecessitate covalent bonding to the substrate. Third, the peptidoglycanlayer is more cross-linked and thick in stationary phase than inlogarithmic phase. Optimization of the method can be performed bytesting MSSA and MRSA strains at lag-phase, logarithmic phase, andstationary phase. To test S. aureus in lag phase, a culture isinoculated (MHB+2% NaCl to a density of ˜10⁷ cfu/ml) and incubated at35° C. for 90 min. The bacteria are harvested by centrifugation, washedonce, and then immobilized on the glass slide surface. The cells shouldbe tested immediately. To test S. aureus in stationary phase, cells arecultured as above, but overnight.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose skilled in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications, and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddo not constitute any admission as to the correctness of the dates orcontents of these documents.

EXAMPLES

Herein is described a method for applying shear stress and/or chemicalstress to bacterial cells to enable rapid detection of antibioticsusceptibility. The purpose of the shear stress is to strain the cells,specifically by damaging the cell wall. Methicillin, as with otherantibiotics in its class, inhibits the biosynthesis of the cell wall.Thus it was hypothesized that damage to the cell wall in the presence ofantibiotics causes cell damage and eventual death in susceptiblestrains, whereas resistant strains will be able to repair themselves.The imposed mechanical strain will shorten the time to detectionsignificantly, and detection may be achieved on the order of minutes totens of minutes. In addition, adding mechanical stress to the bacteriawill also activate other biochemical pathways that are targeted byantibiotics with other mechanisms of action (e.g., other thanbiosynthesis of the cell wall), therefore this methodology is notlimited to antibiotics that act by inhibiting cell wall biosynthesis.

Example 1 Exemplary Device for Determining Antibiotic Susceptibility

Described herein is an exemplary device for determining antibioticsusceptibility/resistance of bacteria. The device comprises a flow cellthat enables controlled shear stress to be applied to immobilizedbacterial cells in the presence of growth media. Fluid will flow throughthe channel, simultaneously applying shear stress to the bacterial cellsand delivering growth media, fluorescent stain, and antibiotic. In someembodiments, the fluorescent stains are chosen such that they do notenter the cells unless the cell wall is permeabilized or the membranepotential changes, and thus will not interfere with cellular repair. Ifthe bacteria are susceptible to the antibiotic, the stress on the cellswill eventually cause the cells to die and thus become fluorescentlystained. If the bacteria are resistant to the antibiotic, they will beable to repair their cell walls in the presence of the antibiotics andtherefore remain unstained.

In one embodiment, the device comprises a flow cell comprised of twoglass slides (one functionalized and one non-functionalized) separatedby a gasket of silicone rubber that defines a single channel (˜500 μmwide). The flow cell design allows standard glass slidefunctionalization methods to be used to immobilize the bacteria to thebase of the flow channel. After bacterial immobilization, the glassslides and silicone rubber are clamped together using a metal housingdesigned to be integrated into the microscope stage. The housing isattached to a syringe pump to control the shear stress on theimmobilized bacteria. The bacteria can be monitored by a microscope withphase/contrast and fluorescence capabilities. The attached camera canacquire images as fast as 10/sec, using e.g., ImageJ (available on theworld wide web at rsbweb.nih.gov/ij/) to analyze the data.

The shear rates in the system were estimated using an ‘infinite’parallel plates (i.e. negligible edge effects) fluid dynamics modelwhich states that the shear rate (du/dx) is

$\frac{\partial u}{\partial x} = \frac{6Q}{{wh}^{2}}$

where Q is the bulk flow rate through the channel, w is the width of thechannel, and h is the height of the channel. With a ˜300 μm wide by 500μm high channel attached to a KDS100 syringe pump (flow rates from 0.1μL/hr to 426 mL/hr), shear rates up to 9,000 s⁻¹ can be achieved.

For the initial prototype system, an Olympus IX81 microscope equippedwith fluorescence and phase/contrast capabilities was used to gatherinitial data and to define any additional system requirements. Thefollowing three additions were added: a motorized microscope stage,integrated temperature control, and a recirculating pump. The stage ofthe microscope is motorized to allow a series of alternating photos of achannel with antibiotics and one without antibiotics. A temperaturecontrol can be incorporated into the device to maintain the bacterialcells in log phase. Initial experiments have demonstrated that arecirculating fluidic design can be used to maintain shear forces over alonger period of time than is allowed with the syringe pump, which needsto be re-filled every 5-10 mins. Fluidics connections can includeindependent loops for re-circulating warmed growth media (up to 40° C.)via a water bath, but maintaining flexibility in the design to allow asingle pass fluidic path if recirculation is not optimal. The fluidicpathway can include syringe junctions for introduction of definedquantities of chemicals (e.g. antibiotics).

The flow cell design can be extended to include multiple channels ofidentical dimensions. In the prototype system, the channels were handcut from a silicone rubber sheet (thickness 230 μm). Although thismethod is convenient for gathering initial data, the manufacturingmethod can be altered to provide defined and reproducible channeldimensions. At least two alternate methods of cutting can be used, whichinclude laser ablation and water jet cutting. UV-nanosecond andIR-femtosecond lasers have also been shown to be effective tools toablate silicone rubber at the channel sizes described herein withoutchemically modifying the material. The alternative solution is water jetcutting which would produce appropriate channels, but the minimumfeature size is 500-1000 μm.

Example 2 Method for Determining Antibiotic SusceptibilityFunctionalization of Slides

Capture agent proteins for immobilizing bacteria can be purchased fromcommercial sources in purified form (Sigma Aldrich) and nonspecificallybound to a glass slide following a basic protocol similar to coatingmicrotiter plates that has been used in other flow cell studies of S.aureus (Shenkman, B., et al., Infect Immun (2001) 69(7):4473-8; Mascari,L., et al., Biotechnol Bioeng (2003) 83(1):65-74; Mascari, L. and J. M.Ross, Ann Biomed Eng (2001) 29(11):956-62; Mohamed, N., et al., InfectImmun (1999) 67(2): 589-94; Brouillette, E., et al., Vaccine (2002)20(17-18):2348-57). Optimal coating conditions can be determinedempirically.

Briefly, the capture agent is resuspended in a buffer (either 0.05phosphate buffer pH 7 or 0.05M carbonate/bicarbonate buffer pH 9.6), anda small volume is placed on the glass slide. The slide is incubated at37° C. in a humid chamber for 1-2 hrs. The slide is rinsed and thenblocked overnight with a blocking agent, such as 1% non-fat milk proteinor BSA. In some embodiments, a glass slide is the preferred bacterialcapture substrate because of the superior optical properties,availability of chemically activated surfaces, substrate flatness,geometric compatibility with standard microscopes, and low cost.However, it is also possible to use fibrinogen or fibronectinimmobilized on polystyrene surfaces.

Several combinations and concentrations of the three capture agents(ranging from 1 to 100 μg/ml) are tested to determine the optimal ratioand concentration for tight binding of S. aureus. As the USA300 straindoes not produce capsule (Montgomery, C. P., et al., J Infect Dis(2008)), it is well suited for initial adherence studies. It carriesgenes encoding staphylococcal adhesins that bind to fibronectin,fibrinogen, and elastin.

Staphylococcus aureus: An Exemplary Antibiotic Resistant Bacterium

Staphylococcus aureus is an exemplary bacterium that exhibits antibioticresistance and is used herein to optimize the methods and devicesdescribed herein. S. aureus was chosen as a model system to test therapid detection of methicillin-resistance because S. aureus is veryclinically relevant, is widespread and methicillin is specific fortargeting cell wall biosynthesis.

1. Antibiotic Resistance of Staphylococcus aureus

The number of S. aureus infections is increasing, as is the resistanceof S. aureus to a variety of antibiotics. Methicillin-resistant Staph.aureus (MRSA) account for 40%-60% of nosocomial S. aureus infections inthe U.S., and many of these strains are multi-drug resistant. Notoriousfor decades as a major source of nosocomial infections, S. aureus hasrecently taken on a new role in causing an escalating number ofcommunity-acquired infections in hosts without significant predisposingrisk factors. Virulent community-acquired MRSA strains are becoming moreprevalent across the U.S. and Europe.

Methicillin resistance in staphylococci is caused by the expression ofPBP2a encoded by the mecA gene that is located on a genetic elementcalled the staphylococcal cassette chromosome (SCC). SCCmec is a groupof mobile DNA elements of 21 to 67 kb that are found integrated into thechromosome of MRSA strains. Unlike hospital-acquired MRSA strains thatcarry large SCCmec elements and show a somewhat slower growth rate thanmethicillin-sensitive strains, the community-acquired MRSA strains oftencarry SCCmec type IV or V elements and show no defect in growth rate.The latter elements are smaller in size than the SCCmec types I, II, andIII found in hospital-acquired MRSA, and their dissemination has beenobserved globally (Baggett, H. C., et al. J Infect Dis, (2004)189(9):1565-73; David, M. D., et al. J Hosp Infect (2006) 64(3):244-50;Gilbert, M., J. CMAJ (2006) 175(2):149-54; Gosbell, I. B., et al.Pathology (2006) 38(3):239-44; Kazakova, S. V., J. C. et al. N Engl JMed (2005) 352(5):468-75). Many of the community-acquired MRSA strainsproduce the Panton-Valentine leukocidin (PVL), and this trait correlateswith increased strain virulence (Baggett, H. C., et al., (2004), supra).PVL-producing S. aureus isolates have been associated with skinabscesses, as well as with necrotizing pneumonia, and often infect youngand previously healthy patients (Baggett, H. C., et al., (2004), supra;Gillet, Y., et al. Lancet (2002) 359(9308):753-9; Muller-Premru, M., etal., Eur J Clin Microbiol Infect Dis (2005) 24(12):848-50).

Not only has resistance to methicillin among S. aureus isolates becomemarkedly more common, but also numerous S. aureus strains with reducedsusceptibility to vancomycin have been reported. Seven clinical isolatesof S. aureus that carry the vanA resistance gene and are fully resistantto vancomycin have been reported. These isolates are also methicillinresistant (MMWR Morb Mortal Wkly Rep, (2002) 51(26); MMWR Morb MortalWkly Rep (2004) 53(15): 322-3; Chang, S., et al. N Engl J Med (2003).Because S. aureus cannot always be controlled by antibiotics and becauseMRSA isolates are becoming increasingly prevalent in the community,additional control strategies are sorely needed.

The mechanism of action of β-lactam antibiotics, such as penicillin andmethicillin, is to inhibit bacterial cell wall (peptidoglycan)biosynthesis. Penicillin binding proteins (PBPs) are membrane boundDD-peptidases that have evolved from serine proteases, and theirbiochemical activity is mechanistically similar (Ghuysen, J. M., TrendsMicrobiol, (1994) 2(10):372-80; Waxman, D. J. and J. L. Strominger, AnnuRev Biochem, (1983). 52: p. 825-69). These enzymes catalyze thetranspeptidation reaction that cross-links the peptidoglycan of thebacterial cell wall. The β-lactam antibiotics are stereochemicallyrelated to D-alanyl-D-alanine (Ghuysen, J. M., Trends Microbiol, (1994)2(10):372-80) and are substrate analogs that covalently bind to the PBPactive-site serine, inactivating the enzyme. PBPs 1, 2, and 3, whichhave high affinity for most β-lactam antibiotics, are essential for cellgrowth and for the survival of susceptible strains, and binding ofβ-lactams by these PBPs is lethal (Chambers, H. F. and M. Sachdeva, JInfect Dis (1990) 161(6):1170-6; Georgopapadakou, N. H., et al.,Antimicrob Agents Chemother (1986) 29(2):333-6). Methicillin-resistancein staphylococci has been termed intrinsic because it is not due to thedestruction of the antibiotic by β-lactamase, but is instead provided bypenicillin-binding protein 2a (PBP2a) (reviewed in (Chambers, H. F.,Clin Microbiol Rev (1997) 10(4):781-91). In methicillin-resistant cells,PBP2a, with its low affinity for binding β-lactam antibiotics (Brown, D.F. and P. E. Reynolds FEBS Lett (1980) 122(2):275-8; Hartman, B. J. andA. Tomasz, J Bacteriol (1984) 158(2):513-6; Utsui, Y. and T. YokotaAntimicrob Agents Chemother (1985) 28(3):397-403; Hayes, M. V., et al.,Antimicrob Agents Chemother (1981) 29:119-122), can substitute for theessential functions of high-affinity PBPs (PBPs 1, 2 and 3) atconcentrations of antibiotic that are otherwise lethal.

2. Immobilization of S. aureus

Staphylococcal adhesion to host tissue is mediated by surface exposedmolecules named adhesins, which specifically bind to host receptors,including fibronectin, fibrinogen, thrombospondin, collagen, elastin,laminin, von Willebrand factor, osteopontin, bone sialoprotein, andvitronectin (Shenkman, B., et al. Infect Immun (2001) 69(7):4473-8;George, N. P., et al., J Infect Dis (2007) 196(4):639-46; George, N. P.,et al., Arterioscler Thromb Vasc Biol (2006) 26(10):2394-400; Mascari,L. and J. M. Ross Ann Biomed Eng (2001) 29(11):956-62; Fallgren, C., etal., Biomaterials (2002) 23(23):4581-9; Herrmann, M., et al., J InfectDis (1997) Herrmann, M., et al., Infect Immun (1991) 59(1):279-88;Herrmann, M., et al., J Infect Dis (1988) 158(4):693-701; Liang, O. D.,et al., J Biochem (1994) 116(2):457-63; Smeltzer, M. S., et al., Gene(1997) 196(1-2):249-59; Brouillette, E., et al., Vaccine (2002)20(17-18):2348-57; Vercellotti, G. M., et al., Am J Pathol (1985)120(1):13-21). A study of the prevalence and chromosomal map location ofS. aureus adhesin genes revealed that the genes for fibronectin—(fnbAand fnbB), fibrinogen—(fib and clfA), and elastin-binding proteins(ebpS) are highly conserved. From a review of the dynamic adhesionstudies with S. aureus, preferred capture agents are fibrinogen andfibronectin. These proteins are especially well-suited forimmobilization of S. aureus to glass slides since fibronectin bindingproteins A and B, as well as clumping factor B (a fibrinogen bindingprotein), are expressed during the logarithmic phase of growth, whereasclumping factor A, the major S. aureus fibrinogen binding protein, issurface-associated in both logarithmic- and stationary-phase cultures.This is important since S. aureus may produce a capsule in thestationary growth phase, and in most strains the clumping factor proteinwill not be masked by the polysaccharide capsule. An additional gene(map) which encodes a broad specificity adhesin (MHC analog protein)that mediates low level binding of several proteins (osteopontin,collagen, bone sialoprotein, vitronectin, fibronectin, and fibrinogen)is also highly conserved and can be used as a capture agent, however itshould be noted that this adhesin mediates only low-level binding andrequires further optimization (see Optimization of Methods sectionherein).

The Staphylococcus aureus (MRSA strain MW2 and MSSA strain Sanger 476)were grown to log phase in Mueller-Hinton broth with 2% sodium chloride(MH2) and then immobilized on epoxide slides, which is a non-specificmethod for immobilizing bacteria and can be used instead of the adhesinmechanism. To immobilize the bacteria, 100 μL of culture was placed onthe epoxide slide (Super-epoxy II, Telechem International) and incubatedat 37° C. for 45 min in a humid environment. The immobilized cells werebriefly rinsed with water and Block-it buffer (Telechem International)to quench any remaining un-reacted epoxide bonds. A series of controlexperiments was conducted including (1) varying protocols forimmobilization to optimize cell density and viability, (2) varying shearrates to confirm it does not lead to significant loss of bacteria off ofslide or due to cell death, and (3) varying concentrations of oxacillinand lysostaphin. In these preliminary studies, a set of conditions wasfound that permits differentiation between MRSA and MSSA strains in lessthan 15 min.

On two separate slides, MSSA (Sanger 476) and MRSA (MW2) strains wereimmobilized. The cells were assembled in channels of ˜300 μm width andstained with 0.5 μM Sytox green in MH2. The immobilized cells werestressed with 0.6 ng/mL lysostaphin, ˜4200 s⁻¹ shear rate, and 10 μg/mLoxacillin for 15 mins after a 40 min pretreatment with lysostaphin andshear alone. The lysostaphin concentration was chosen to be lower thanthe MIC (˜1 ng/mL). The oxacillin concentration is greater than the MICfor the MSSA (0.5 μg/mL) and less than that for the MRSA (>16 μg/mL).More MSSA cells died during the course of the experiment than MRSAcells.

Strains of Staphylococcus useful for optimizing the methods describedherein include several MRSA strains whose genomes have been sequenced:

(a) USA300 strain NRS384; highly virulent and has spread across the U.S.and other countries causing community-acquired staphylococcal infections(primarily skin and soft tissue infections). USA300 carries a type IVSCCmec element but does not make a capsule.

(b) Strain MRSA252; belongs to the clinically important EMRSA-16 clonethat is responsible for half of all MRSA infections in the U.K. and isone of the major MRSA clones found in the U.S. (USA200). MRSA252 carriesa type II SCCmec element and, produces a serotype 8 capsule.

(c) Strain MW2; belongs to the USA400 group. It carries a type IV SCCmecelement, produces the Panton-Valentine leukocidin, and is associatedwith skin and soft tissue infections, as well as necrotizing pneumoniain children. USA400 was the most widespread community-acquired S. aureusstrain prior to 2001 when USA300 emerged in the U.S. population. Thisstrain also produces a type 8 capsule;

(d) A USA100 strain; represents the predominant U.S. hospital-acquiredMRSA strain (also known as the New York/Japan clone). A useful strain ofUSA100 is strain NRS382 (McDougal, L. K., et al., J Clin Microbiol(2003) 41(11):5113-20). NRS392 belongs to sequence type (ST) 5, and thusit is closely related to S. aureus strains N315 and Mu50 (both of thesestrains have been sequenced).

In addition, the following methicillin-sensitive Staphylococcus aureus(MSSA) strains can be used as a control for antibiotic susceptibility:

(a) MSSA476; a representative of an invasive community-acquired MSSAclone that was isolated from a 9-year old child with no predisposingrisk factors who developed osteomyelitis and bacteremia; strain MSSA476is a member of the USA400 pulsotype and is capsule negative;

(b) Strain Newman is a well-characterized MSSA strain whose genome wasrecently sequenced. Strain Newman is genetically very similar to theMSSA strain NCTC8325 and the MRSA strain COL, and it produces a serotype5 capsule. Newman is a better choice than strain NCTC8325 because thelatter carries a mutation in the rsbU locus that renders the straindeficient in sigma B regulated genes; and

(c) MSSA USA300 strain.

Lysostaphin Susceptibility of MW2 and Sanger 476

Described herein is experimental evidence that the methods and devicesdescribed herein differentiate between a methicillin-resistant strain(MW2) and a methicillin-susceptible strain (Sanger 476) ofStaphylococcus aureus in the presence of shear stress and chemicalstress (lysostaphin with or without oxacillin). The difference wasdetectable within 15 mins of introducing oxacillin. In this experiment,lysostaphin was used to increase the stress on the Staphylococcus cellsabove that imposed by the fluidic shear. In the interpretation of theseresults it is important to understand the sensitivity of the two strainsto lysostaphin. As published values were not readily available, thelysostaphin susceptibility of MW2 and Sanger 476 was empiricallydetermined.

A classical microdilution broth method was used to determine thelysostaphin minimum inhibitory concentrations for strains MW2 and Sanger476 (Jones, R. N., et al. Manual of clinical microbiology, A. Balows, J.W. J. Hausler, and H. J. Shadomy, Editors. 1985, American Society forMicrobiology: Washington D.C. p. 972-977). Briefly, logarithmic-phasebacteria were cultivated in Mueller-Hinton cation-adjusted broth (MHB)and adjusted to a concentration of 1×10⁶ CFU/mL. Sterile microtiterplates containing 100 μl of lysostaphin diluted in MHB were inoculatedwith 100 μl of the bacterial suspension to yield 5×10⁵ CFU/mL. Afterovernight incubation at 37° C. overnight, the MIC for both strains was0.06 μg/mL.

As the sensitivity to lysostaphin is the same for both strains, theresults presented herein strongly demonstrate that shear stress can beused to rapidly determine antibiotic susceptibility.

Detecting Mechanical Strain on Bacterial Cells with DiBAC₄(3)

Studies with Sytox Green were successful in that the stain is verybright for dead cells with a low background of staining for the livecells. Since Sytox green works by staining the nucleic acids, it iseffective mainly at the late stage of antibiotic action (when the cellsare truly dead). Thus, to measure the antibiotic susceptibility in thesystem described herein with Sytox Green within tens of minutes, oneneeds to apply very high shear rates and additional chemical stress(lysostaphin).

On the other hand, DiBAC₄(3) is a stain that measures changes inmembrane potential and is therefore a more sensitive and earlierindicator of cellular damage. It was recently confirmed that DiBAC₄(3)does stain cells under high stress when Sytox green does not stain them.As such, one can achieve rapid detection of antibiotic susceptibilitywith mechanical stress alone (i.e. no lysostaphin) when using DiBAC₄(3)as the stain.

Example 3 Exemplary 4-Channel Flow-Cell

Also provided herein is an exemplary flow cell comprising four channelsarranged in a parallel manner as shown herein in FIG. 8A. The flow cellcan be used in conjunction with the instrument and software depicted inFIG. 8B.

This 4-channel arrangement was used to test a single strain of bacteriain the presence or absence of chemical stress (e.g., lysostaphin). Theflow rate used in the channels was 1 mL/min and 0.5 μM Sytox Green wasused to stain damaged or dying cells.

FIG. 9A shows data using a Sanger strain of staphylococcus that issusceptible to oxacillin treatment, while FIG. 9B shows data using amethicillin and oxacillin resistant strain of bacteria (MW2). In eachexperiment, bacteria in two channels were exposed to 0.7 ng/mLlysostaphin (lyso) in the presence or absence of the antibioticoxacillin (oxa; 50 μg/mL). The four experimental groups are as follows:(i) control (no oxa; no lyso), (ii) lyso only; (iii) oxa only, and (iv)lyso and oxa together.

The level of fluorescence in Sanger cells in the channel treated withlyso alone was not substantially different from the level offluorescence in the control channel. Fluorescence was increased at atime point of approximately 20 min in both the lyso/oxa treated Sangergroup as well as the Sanger cells treated with oxa alone. However, theSanger cells in the channel treated with both lyso and oxa died fasterthan the Sanger cells receiving oxa alone. These data indicate thatchemical stress can be used to augment mechanical shear stress andincrease the susceptibility of bacteria to antibiotic induced damage inan assay setting. Furthermore, these data demonstrate successful use ofthe methods and devices described herein.

FIG. 9B shows that there was no substantial increase in fluorescence ofMW2 cells treated with oxacillin compared to control MW2 cells. Thisresult is expected since the MW2 strain is resistant tooxacillin-mediated cell death. MW2 cells treated with lyso alone showeda small increase in fluorescence further confirming that chemical stresscan be used successfully in a variety of bacterial strains with themethods and devices described herein.

The 4-channel system can also be used to test two bacterial strainssimultaneously as shown herein in FIGS. 10A and 10B.

1. (canceled)
 2. A method for determining sensitivity of bacteria to anantibiotic, the method comprising: (a) immobilizing the bacteria from abacterial suspension by covalent attachment to a solid support, (b)contacting said immobilized bacteria with an agent comprising a reportermoiety, which preferentially binds to damaged bacterial cells, (c)subjecting said immobilized bacteria to a stressor in the presence of anantibiotic without the need for bacterial growth phase, (d) detecting asignal from said reporter moiety in said immobilized bacteria aftercontacting said covalently immobilized bacteria with the stressor in thepresence of the antibiotic and without the need for bacterial growthphase, and (e) comparing the signal from said immobilized bacteria inthe presence of the antibiotic to a control comprising immobilizedbacteria subjected to the stressor in the absence of the antibiotic,wherein detection of an increase in said signal in the presence of anantibiotic compared to a control indicates that said bacteria aresusceptible to said antibiotic, and wherein a signal that is comparableto the control in the presence of an antibiotic indicates that saidbacteria are resistant to said antibiotic.
 3. The method of claim 2,wherein said detecting step comprises detecting fluorescence.
 4. Themethod of claim 2, wherein said reporter moiety comprises a fluorescentdye.
 5. The method of claim 4, wherein said fluorescent dye detects gramnegative and/or gram positive bacteria.
 6. The method of claim 4,wherein said fluorescent dye is SYTOX green.
 7. The method of claim 4,wherein said fluorescent dye is DiBAC₄(3).
 8. The method of claim 2,wherein said stressor comprises physical stress.
 9. The method of claim8, wherein the physical stress comprises shear stress, osmotic stress,acidic pH or basic pH.
 10. The method of claim 2, wherein said stressorcomprises chemical stress.
 11. The method of claim 10, wherein saidchemical stressor comprises lysostaphin, an endolysin, lysozyme,oxidative stress or a porin.
 12. The method of claim 2, wherein saidstressor comprises physical and chemical stress.
 13. The method of claim1, wherein said bacteria are pathogenic bacteria.
 14. The method ofclaim 13, wherein said pathogenic bacteria are capable of infecting andcausing disease in a human host.
 15. The method of claim 2, wherein thesolid support is glass.
 16. The method of claim 2, wherein the solidsupport is comprised in a microfluidic channel.
 17. The method of claim16, comprising multiple microfluidic channels.
 18. The method of claim17, wherein each of the multiple microfluidic channels is exposed to adifferent antibiotic agent.
 19. The method of claim 18, wherein each ofthe multiple microfluidic channels is exposed to a differentconcentration of the antibiotic.
 20. The method of claim 2, wherein theantibiotic comprises a combination of antibiotics.
 21. The method ofclaim 2, wherein said method comprises a high-throughput assay.